Investigating the Effects of N2-Acetylphenelzine in the Sexes in Experimental Autoimmune
Encephalomyelitis
by
Emma Frieser
A thesis submitted in partial fulfillment of the requirements for the degree of
Master of Science
Neuroscience and Mental Health Institute
University of Alberta
© Emma Frieser, 2017
N2-Ac-PLZ, SEX DIFFERENCES & EAE ii
Abstract
Chronic pain is a highly prevalent symptom in Multiple Sclerosis (MS) and affects
approximately half of patients at some stage of the disease. MS affects women more frequently
than men and neuropathic pain is also reported as more severe, and frequent, in females with the
disease. The underlying causes of neuropathic pain remain to be elucidated and it is unclear why
females are more greatly affected. The animal model experimental autoimmune
encephalomyelitis (EAE) is used to study the pathophysiology of MS and MS-related pain.
Studies have shown that pain behaviours and the disease course in mice with EAE may be
improved with the administration of the antidepressant phenelzine (PLZ) which acts to elevate
noradrenaline (NA), serotonin (5-HT), and gamma-aminobutyric acid (GABA) levels.
Interestingly, studies have also demonstrated significant sex differences in the behavioural
responses to PLZ and an acetylated derivative, N2-acetylphenelzine (N2-Ac-PLZ) (that elevates
NA and 5-HT, but not GABA, levels) in the formalin pain assay. We have found that female
mice do not respond to N2-Ac-PLZ in this particular pain model. As such, I sought to examine
whether N2-Ac-PLZ beneficially improves pain behaviours and disease course within the sexes
in mice with EAE.
C57/BL6 mice of both sexes were treated with myelin oligodendrocytes glycoprotein
35-55 (MOG35-55) in Complete Freund’s Adjuvant (CFA) and pertussis toxin to induce EAE. The
mice were monitored daily, and starting on day 7 post-induction, chronically treated with a
vehicle or N2-Ac-PLZ every other day until day 34 post-induction. I used behavioural tasks to
assess exploratory, anxiety-like, and mechanical hypersensitivity behaviours in the mice, as well
as high performance liquid chromatography (HPLC) to assess spinal levels of NA, 5-HT, and
GABA.
N2-Ac-PLZ, SEX DIFFERENCES & EAE iii
In contrast to the formalin model, I find that N2-Ac-PLZ improved tactile hypersensitivity only
in female mice with EAE. N2-Ac-PLZ treatment had no effect on the disease course of female or
male mice with EAE. My examination of neurotransmitter levels within the spinal cord revealed
that N2-Ac-PLZ treatment acted to increase levels of NA and 5-HT in both females and males,
but with higher elevations observed in females.
This study shows that treatment with N2-Ac-PLZ can attenuate tactile hypersensitivity
in a disease-related chronic pain paradigm in a sex-specific manner. These data provide further
evidence of sex differences in EAE, and insight into the causes of pain in the disease.
N2-Ac-PLZ, SEX DIFFERENCES & EAE iv
Preface
This research was conducted in collaboration with Dr. Bradley Kerr and Dr. Glen
Baker at the University of Alberta. Dr. Kerr’s lab induced the mouse model of EAE used in the
study. After the mice were euthanized and tissue was removed, all samples were processed with
high-performance liquid chromatography (HPLC) in co-operation with Dr. Baker’s lab at the
University of Alberta where I conducted my work. My work included clinical monitoring,
behavioural measures, tissue processing, all HPLC, and subsequent data analysis. The thesis was
written entirely by myself, with edits suggested by an examining committee. The project was
conducted under ethical approval of the University of Alberta Animal Research Ethics Board.
N2-Ac-PLZ, SEX DIFFERENCES & EAE v
Acknowledgments
First and foremost, I want to thank my supervisor Dr. Bradley Kerr, for taking a chance
on a psychology student with little knowledge of Neuroscience. Your compassion and constant
motivation were a blessing through the toughest times of my degree. Thank you for being the
type of mentor a grad student could only dream to have. Thank you to my colleagues past and
present: Kevin, Saad, Ana, Curtis, Kasia, Liam, Gustavo, and especially Katherine, who
managed to guide me through the last two years by imparting her vast wisdom and somehow
maintaining my (relative) saneness. Being in the Kerr Lab was an incredibly wonderful way to
spend the last two years, and I am so grateful to everyone in it.
Additionally, I would like to thank Dr. Glen Baker and Dr. Peter Smith of my
supervisory committee for offering their knowledge and guidance throughout the entirety of my
thesis. Thank you to Dr. Baker and Gail Rauw in the Neurochemical Research Unit who played a
large role in my thesis project; Gail, your immense knowledge and kindness has not gone
unnoted -- thank you so much for all your help.
Thank you to my parents, Keith and Mairia, for their unwavering love, support, and
understanding in my pursuit of higher education. I am so grateful to have been provided with the
opportunities to pursue these dreams, and I love you both so much. Thank you to my brother and
sister, Bowen and Jesse, for your steady encouragement and bragging; you are both such an
inspiration to me, and I am thankful to have you in my life. Lastly, I would like to thank my
lifelong friends for being a steady source of happiness and laughter.
I truly appreciate all you have done for me;
this thesis is a reflection of the love and support I’ve received.
N2-Ac-PLZ, SEX DIFFERENCES & EAE vi
Table of Contents
Page
Abstract ii
Preface iv
Acknowledgements v
Table of Contents vi
List of Tables ix
List of Figures x
Glossary of Terms xi
1. Introduction 1
1.1 MS & Sex Differences 1
1.2 EAE & Pain 2
1.3 Antidepressants in Pain 3
1.4 NA 5
1.5 5-HT 6
1.6 GABA 7
1.7 Phenelzine (PLZ) 9
1.8 N2-Acetylphenelzine (N2-Ac-PLZ) 11
1.9 Formalin: Effects of PLZ & N2-Ac-PLZ 11
1.10 Present Study 12
2. Methods 14
2.1 Subjects and Experimental Treatments 14
2.1-1 Naïve N2-Ac-PLZ Experiment 14
N2-Ac-PLZ, SEX DIFFERENCES & EAE vii
2.1-1.1 Subjects and Treatment 14
2.1-1.2 Behaviour 14
2.1-1.2.1 Exploratory Behaviour 15
2.1-1.2.2 Anxiety 15
2.1-1.2.3 Mechanical Hypersensitivity 15
2.1-1.2.4 Motor Function 16
2.1-1.3 Tissue Collection 16
2.1-1.4 HPLC 16
2.1-1.5 Statistics 17
2.1-2 Disease Related Chronic Pain Experiment 18
2.1-2.1 Subjects, Treatment and EAE Induction 18
2.1-2.2 Behaviour 19
2.1-2.2.1 Exploratory Behaviour 19
2.1-2.2.2 Mechanical Hypersensitivity 19
2.1-2.2.3 Motor Function 19
2.1-2.3 Tissue Collection, HPLC, Statistics 19
3. Results 20
3.1 Effects of N2-Ac-PLZ in Naïve Mice 20
3.1-1 Exploratory Behaviour 20
3.1-2 Anxiety Behaviour 21
3.1-3 Mechanical Hypersensitivity 22
3.1-4 Motor Function 22
3.1-5 Neurotransmitter Levels 22
N2-Ac-PLZ, SEX DIFFERENCES & EAE viii
3.2 Effects of N2-Ac-PLZ in Mice with EAE 23
3.2-1 Disease Course and Day to Onset 23
3.2-2 Exploratory Behaviour 24
3.2-3 Mechanical Hypersensitivity 25
3.2-4 Motor Function 25
3.2-5 Neurotransmitter Levels 25
3.3 Figures 27
4. Discussion 38
4.1 The Present Study 38
4.2 Disease Course 38
4.3 Exploratory and Anxiety Like Behaviour 39
4.4 Pain Behaviours: Mechanical Hypersensitivity 39
4.5 Neurotransmitter Levels 40
4.6 Mechanism of Action 41
4.6-1 Immune Response 42
4.6-2 Oxidative Stress & Pain 44
4.7 Decreases in GABA levels 46
Conclusion 46
References 48
N2-Ac-PLZ, SEX DIFFERENCES & EAE ix
List of Tables
Table 1. Peak clinical scores in female and male mice with EAE. 32
N2-Ac-PLZ, SEX DIFFERENCES & EAE x
List of Figures
Figure 1. Noradrenaline (NA) molecular structure. 6
Figure 2. Serotonin (5-hydroxytryptamine) molecular structure. 6
Figure 3. Gamma-aminobutyric acid (GABA) molecular structure. 8
Figure 4. How PLZ acts to increase levels of NA, 5-HT, and GABA. 9
Figure 5. N2-Ac-PLZ structure. 11
Figure 6. Naïve exploratory behaviour measured using the open field test. 27
Figure 7. Naïve anxiety behaviour measures using the elevated plus maze (EPM). 28
Figure 8. Von-Frey hairs used to measure mechanical hypersensitivity in naïve mice. 29
Figure 9. Naïve mice tested on the rota-rod as a locomotor control measure. 30
Figure 10. Spinal levels of NA, 5-HT, and GABA in naïve mice were measured using high
performance liquid chromatography (HPLC). 31
Figure 11. The effects of N2-Ac-PLZ on clinical scores of EAE mice. 33
Figure 12. Exploratory behaviour in mice with EAE measured using the open field test. 34
Figure 13. Von-Frey hairs measured mechanical hypersensitivity in mice with EAE. 35
Figure 14. Mice with EAE tested on the rota-rod as a locomotor control measure. 36
Figure 15. Spinal levels of NA, 5-HT, and GABA in mice with EAE measured with HPLC. 37
N2-Ac-PLZ, SEX DIFFERENCES & EAE xi
Glossary of Terms & Abbreviations
Multiple Sclerosis: MS
Experimental Autoimmune Encephalomyelitis: EAE
Noradrenaline: NA
Serotonin: 5-HT
Gamma-aminobutyric acid: GABA
Myelin Oligodendrocytes Glycoprotein: MOG
Complete Freund’s Adjuvant: CFA
Von-Frey Hairs: VF
Central Nervous System: CNS
Phenelzine: PLZ
N2-Acetylphenelzine: N2-Ac-PLZ
Elevated Plus Maze: EPM
High Performance Liquid Chromatography: HPLC
N2-Ac-PLZ, SEX DIFFERENCES & EAE 1
1. Introduction
Chronic pain is a prevalent disorder that can be difficult to diagnose due to varying
definitions. It can be characterized as any pain lasting longer than three months (Elliott, Smith,
Penny, Smith & Chambers, 1999). Up to 30% of people are reported to suffer from chronic pain,
but prevalence rates range widely due to variations in the diagnostic criteria (Benson et al., 2015;
Breivik, Collett, Ventafridda, Cohen & Gallacher, 2006; Dansie & Turk, 2013; Elliott et al.,
1999). Chronic pain is a primary disorder in itself but is also a secondary symptom of primary
autoimmune diseases such as Rheumatoid Arthritis and Multiple Sclerosis (MS) (Mifflin & Kerr,
2016; Pöllmann & Feneberg, 2008). Many MS patients report experiencing chronic pain, such as
headaches and back pain, at some point in their disease course (Pöllmann & Feneberg, 2008).
1.1 MS & Sex Differences
The incidence of MS is much higher in females, who also report higher levels of
neuropathic pain, which is defined as “pain caused by a lesion or disease of the somatosensory
system” (Merskey & Bogduk, 2011; Orton et al., 2006). MS is a neurodegenerative autoimmune
disease that causes significant decline in cognitive, motor, sensory, and affective domains
(Compston & Coles, 2008). The pathophysiology includes white matter abnormalities that can be
detected by magnetic resonance imaging (MRI), demyelination, and axonal degeneration, all of
which can contribute to the symptoms of MS. Chronic pain is a prevalent, and debilitating,
symptom of MS that is reported to affect approximately 43% of patients at some point in their
disease (Solaro et al., 2004). The appearance of MS is heterogeneous, with patients presenting as
relapsing remitting (RRMS), progressive (PMS), or progressive relapsing MS as outlined by the
Multiple Sclerosis Society of Canada (MSSC). This heterogeneity within the clinical population
makes the treatment of MS all the more difficult, since not all patients will respond to available
N2-Ac-PLZ, SEX DIFFERENCES & EAE 2
treatments. Many sex differences have been reported in MS, but the main one is that females
have a higher incidence of MS, as well as reported pain (Bove & Chitnis, 2014; Orton et al.,
2006). There have been multiple theories as to why females are affected more than males, such
as the presence of reproductive hormones, but the mechanisms underlying sex differences in MS
remain to be revealed (Bove & Chitnis, 2014). Heterogeneity and sex differences within the
clinical population are important factors when considering the development and testing of new
treatments in order to more efficiently treat the disease or the pain associated with it (Solaro,
Trabucco & Messmer Uccelli, 2013).
1.2 Experimental Autoimmune Encephalomyelitis (EAE) & Pain
The animal model most commonly used to study MS, experimental autoimmune
encephalomyelitis (EAE), is used in order to investigate the underlying mechanisms of pain in
MS, as well as to develop new treatments (Dray, 1995). EAE is a disease model that mimics both
the pathophysiology and symptoms of pain hypersensitivity in MS, which allows researchers to
effectively test and improve treatments that could beneficially affect the clinical population
(Aharoni, 2013; Olechowski, Truong & Kerr, 2009). There are multiple ways to induce EAE, but
the Kerr laboratory works solely with myelin oligodendrocytes glycoprotein 35-55 (MOG35-55) in
Complete Freund’s Adjuvant (CFA), and pertussis toxin (PT) (Benson, Wong, Tenorio, Baker &
Kerr, 2013). The MOG35-55 and CFA are administered via subcutaneous injections which are
followed by two intraperitoneal injections of PT (Benson et al., 2013; Bittner, Afzali, Wiendl &
Meuth, 2014; Hofstetter, Shive & Forsthuber, 2002). PT facilitates EAE induction and increases
disease incidence and severity, primarily through its ability to facilitate the break-down of the
blood-brain barrier (BBB) (Linthicum, Munoz & Blaskett, 1982). The addition of CFA alongside
PT acts to accelerate the animals’ immune response to the mildly foreign MOG peptide, which is
N2-Ac-PLZ, SEX DIFFERENCES & EAE 3
similar to the mouse’s myelin peptide, and is attacked by their immune system. This specific
model of EAE mimics a chronic progressive disease course often seen in MS patients (Robinson,
Harp, Noronha & Miller, 2014).
Neuropathic pain can be measured in EAE by using standard behavioural assays to
assess sensitivity to mechanical and thermal stimuli (Olechowski et al., 2009). At disease onset
(the first day that mice show clinical signs of EAE; clinical grade 1), thresholds to Von Frey
(VF) hairs were reduced, indicating an increased sensitivity to mechanical stimuli. Mice with
EAE also exhibit prolonged responses following the application of innocuous acetone to the hind
paws in the acetone test (Olechowski et al., 2009; Potter et al., 2016). Olechowski and colleagues
(2009) also determined immunohistologically that mice with EAE who demonstrated enhanced
nociceptive behaviors at disease onset had increased inflammation in the superficial dorsal horn
of the spinal cord including the presence of CD3+ T-cells and reactive gliosis (enhanced
expression of the F4/80 antigen on microglia/macrophages).
Neurotransmitters like noradrenaline (NA), serotonin (5-HT), and gamma-
aminobutyric acid (GABA), play an important role in the development and regulation of pain.
EAE has been shown to decrease levels of all three of these neurotransmitters in the spinal cord
(Musgrave et al., 2011; Musgrave, Tenorio, Rauw, Baker & Kerr, 2011). These findings coincide
with decreased levels of these neurotransmitters in MS, and could possibly be related to the
disease progression and enhanced nociceptive sensitivity seen in EAE. EAE provides a starting
point for researchers to examine the mechanisms of MS and the associated neuropathic pain.
1.3 Antidepressants in Pain
The first line of treatment for neuropathic pain are antidepressants, predominantly
tricyclic antidepressants (TCA’s) and selective norepinephrine-serotonin reuptake inhibitors
N2-Ac-PLZ, SEX DIFFERENCES & EAE 4
(SNRI’s), although occasionally selective serotonin reuptake inhibitors (SSRI’s) are prescribed
(Ardid et al., 2001; Attal et al., 2010; Dworkin et al., 2007; Mika, Zychowska, Makuch,
Rojewska & Przewlocka, 2013). TCA’s, such as amitriptyline and desipramine, modulate
noradrenergic and/or serotonergic function (both in the case of amitriptyline and primarily NA in
the case of desipramine) by inhibiting reuptake of NA and 5-HT into nerve terminals (Mika et
al., 2013; Sindrup, Otto, Finnerup & Jensen, 2005). These drugs produce a number of unpleasant
side effects such as dry mouth, blurred vision, and weight gain, and overdosing with them can
result in cardiotoxicity (Mika et al., 2013; Sindrup et al., 2005; Smith, 1998). SNRI’s, such as
venlafaxine, are more frequently prescribed for the treatment of neuropathic pain than SSRI’s
due to their superior analgesic effects over SSRI’s (Mika et al., 2013; Smith, 1998). The
difference in efficacy between SNRI’s and SSRI’s suggests that the noradrenergic system plays a
significant role in pain (Gendreau et al., 2005; Marks et al., 2009; Stahl, Grady, Moret & Briley,
2005). Another class of antidepressants, the monoamine oxidase inhibitors (MAOI’s), are used
when other avenues such as SNRIs and SSRIs have failed. The MAOIs are a class of
antidepressants that inhibit monoamine oxidases thus, preventing the breakdown of monoamines
and leading to increased levels of NA and 5-HT in the CNS (Dworkin et al., 2007). Phenelzine
is unique in that it also increases brain levels of GABA (Sowa, Holt, Todd & Baker, 2004).
It has been suggested that the therapeutic effect of antidepressants comes from their
ability to increase levels of NA and 5-HT in the synaptic cleft, but increased GABA has been
implicated in the efficacy of antidepressants as well (Attal et al., 2010; Benson et al., 2015;
Mico´, Ardid, Berrocoso & Eschalier, 2006; Dupuis et al., 2016; Eide & Hole, 1993; Mika et al.,
2013; Ortega, Fernández-Pastor, Callado & Meana, 2010). The relationship between chronic
pain and levels of NA, 5-HT, and GABA is not fully understood, but all three individual
N2-Ac-PLZ, SEX DIFFERENCES & EAE 5
neurotransmitters, and their interactions, have been implicated in the development and regulation
of pain. Further research must be conducted to determine why increasing levels of these
neurotransmitters through antidepressant treatment benefits patients experiencing pain.
1.4 NA
NA is a prominent neurotransmitter that is synthesized from dopamine, and plays a
role in the regulation of pain (Pertovaara, 2006; Pertovaara, 2013). The noradrenergic system
spreads extensively throughout the nervous system, with the primary source for ascending and
descending central nervous system (CNS) projections coming from the locus coeruleus (LC), as
well as postganglionic nerve fibers in the periphery (Pertovaara, 2013). Ascending projections
are implicated for the regulation of vigilance, attention, and other cognitive functions. This is
mainly mediated via the dorsal and ventral bundles and periventricular bundle (Cooper, Bloom &
Roth, 2003). Descending projections on the other hand are associated with the control of spinal
motor output. The noradrenergic system also provides strong, primarily inhibitory, innervation to
the spinal dorsal horns, an area that is essential in the pain pathway (Cooper et al., 2003;
Pertovaara, 2013; Sawynok, 2003; Westlund & Coulter, 1980). An abundance of noradrenergic
terminals in the spinal dorsal horn supports the idea that noradrenergic descending projections
from the LC play an important role in regulating different aspects of somatomotor and autonomic
function (Westlund & Coulter, 1980). This extensive innervation of the superficial dorsal horn
by the noradrenergic system is proposed to be pain inhibitory, with the LC being considered a
“pain suppressor” (Pertovaara, 2013). NA mediates its effects through its two catecholamine
receptors, - and -adrenoceptors (Pertovaara, 2013; Ruffolo & Hieble, 1994). Subtypes of -
and -adrenoceptors mediate different effects based on their location. -Adrenoceptors (1A,
1B, 1D, 2A, 2B, 2C) are the primary modulators of pain and are located throughout the spinal
N2-Ac-PLZ, SEX DIFFERENCES & EAE 6
Figure 1. Molecular structure of noradrenaline (NA).
cord, though predominantly within the dorsal horn (Nalepa et al., 2005; Ruffolo & Hieble, 1994;
Shi, Winzer-Serhan, Leslie & Hökfelt, 1999). 2-Adrenoceptors are the main noradrenergic
system mediators of pain suppression within the spinal dorsal horn (Ruffolo & Hieble, 1994;
Yaksh & Malmberg, 1994).
1.5 5-HT
Another important neurotransmitter involved in pain processing and modulation is 5-
HT, which is derived from tryptophan and produced in high levels in the raphe nuclei (Eide &
Hole, 1993; Stamford, 1995). 5-HT plays a significant role in the regulation of pain and
nociception within the CNS and is consistently observed to have antinociceptive effects (Dupuis
et al., 2016; Eide & Hole, 1993; Sommer, 2004). The function 5-HT plays in pain is not fully
understood, but it is suggested to contribute to inhibitory descending analgesia that projects to
many areas, including regions of the spinal cord (Stamford, 1995). Yaksh and Wilson (1979)
Figure 2. Molecular structure of serotonin (5-HT).
N2-Ac-PLZ, SEX DIFFERENCES & EAE 7
reported significant analgesic effects of 5-HT, which acted to decrease nociceptive behaviour in
rats. 5-HT may also have effects outside the CNS, but in these peripheral locations it functions as
an inflammatory pain mediator (Dray, 1995). The complexity of 5-HT’s role is likely due to the
variety of receptor subtypes within the CNS (Eide & Hole, 1993). 5-HT receptor subtypes
include 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6, and 5-HT7, several of which play different
roles in pain regulation, such as promoting inhibition within the dorsal horn (Eide & Hole, 1993;
Liu, Su & Lin, 1988; Peng, Lin & Willis, 1996). Activation of the 5-HT2A or 5-HT5A receptor
has been reported to mediate analgesic effects in pain conditions through inhibitory signalling
cascades. Activation of these receptors provides relief from craniofacial and peripheral
hyperalgesia (Dupuis et al., 2016; Hannon & Hoyer, 2008; Millan, 1995; Muñoz-Islas et al.,
2014; Okamoto et al., 2007). In addition, the SSRI fluoxetine is proposed to provide pain relief
via increased activity of 5-HT2A receptors (Anjaneyulu & Chopra, 2004; Dupuis et al., 2016).
Dupuis et al. (2016) also showed that dysfunctional 5-HT2A receptors were implicated in
neuropathic pain, supporting the idea of their involvement in the inhibitory actions of 5-HT in
pain processing. Furthermore, 5-HT2A receptor activation in conjunction with 5-HT1A
antagonism (acting to increase the onset of action) has been associated with the beneficial effects
of antidepressants, which act to normalize inhibitory activity in the bulbospinal pathway in pain
conditions (Ardid et al., 2001; Ardid et al., 1995; Dupuis et al., 2016; Mico´, Ardid, Berrocoso &
Eschalier, 2006).
1.6 GABA
GABA is an abundant inhibitory neurotransmitter within the CNS and is synthesized
from glutamate (GLU), and important excitatory neurotransmitter (Watanabe, Maemura,
Kanbara, Tamayama & Hayasaki, 2002; Zeilhofer, Möhler & Lio, 2009). Synthesis of GABA
N2-Ac-PLZ, SEX DIFFERENCES & EAE 8
from GLU occurs via a decarboxylation reaction that is catalyzed by glutamate decarboxylase
(GAD) (Watanabe et al., 2002). GABA is catabolized by GABA transaminase (GABA-T), which
causes the degradation of GABA, and producing GLU as a bi-product, that is recycled for future
GABA synthesis (Watanabe et al., 2002). GABA’s role in pain production and modulation is not
fully understood, but has been suggested to largely control the communication of nociceptive
signaling from the periphery through the dorsal spinal cord and up to higher brain structures
(Melzack & Wall, 1965). This function is thought to be through inhibitory GABAergic
interneurons within the spinal dorsal horn, an important area implicated in pain (Melzack &
Wall, 1965; Zeilhofer, 2008; Zeilhofer, Möhler & Lio, 2009). GABA serves to maintain
inhibition within the CNS, therefore preventing innocuous stimuli from exciting neurons
Figure 3. Molecular structure of gamma-aminobutyric acid (GABA)
associated with pain production (Zeilhofer, Möhler & Lio, 2009). GABAA, GABAB, and
GABAC receptors mediate these effects, through a range of mechanisms (Chebib & Johnston,
1999). GABAA receptors are more widely distributed throughout the CNS and are thought to be
the primary mediators of pain inhibition (Bormann, 2000; Bormann & Feigenspan, 1995; Jasmin,
Wu & Ohara, 2004; Zeilhofer, Möhler & Lio, 2009). The blockade of GABAA receptors in the
spinal cord produces severe pain hypersensitivity in animal models, and enhanced activity of
GABAA receptors alleviates pain in both animals and humans (Enna & McCarson, 2006; Ugarte,
Homanics, Firestone & Hammond, 2000; Yaksh, 1989; Zeilhofer, Möhler & Lio, 2009).
Reduced levels of GABA within the spinal dorsal horn has been identified as a hallmark of pain,
and effective treatments have been shown to increase GABA levels, resulting in analgesic effects
N2-Ac-PLZ, SEX DIFFERENCES & EAE 9
(Jasmin, Rabkin, Granato, Boudah & Ohara, 2003; Zeilhofer, Möhler & Lio, 2009).
Interestingly, data have suggested that GABA is also released when 5-HT2A receptors are
activated, supporting the idea that both neurotransmitters play critical roles individually as well
as having important interactions with one another to mediate analgesia (Dupuis et al., 2016). The
role of GABA in pain processing and modulation is complex and remains to be fully elucidated,
but overall its function seems to be one of inhibitory pain modulation (Jasmin et al., 2003).
Chronic pain is commonly treated with antidepressants that act to increase functional
availability of NA, 5-HT, and GABA within the CNS (Dworkin et al., 2007). It is important to
acknowledge that although each neurotransmitter plays their own individual roles in pain
production and modulation, the interactions between them are also important to consider when
studying the mechanisms underlying chronic pain.
1.7 Phenelzine (PLZ)
Phenelzine (PLZ) is a non-selective, irreversible MAOI that produces long-lasting
increases in NA and 5-HT. Through the production of the active metabolite β-
phenylethylidenehydrazine (PEH) it can elevate GABA levels via the inhibition of the enzyme
Figure 4. PLZ and its active metabolite PEH act to increase levels of NA, 5-HT, and GABA.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 10
GABA-T (Mackenzie et al., 2010; Parent, Habib & Baker, 2000). PLZ is classified as a non-
selective MAOI because it acts to inhibit both isoforms, MAO-A and MAO-B. MAO-A shows a
higher affinity for NA, 5-HT, and Da, whereas MAO-B associates with PEA and deprenyl
(Johnston, 1968; Knoll & Magyar, 1972; Shih & Thompson, 1999). PLZ treatment has been used
for a variety of disorders, although primarily for depression and anxiety. Its anxiolytic effects can
be observed clinically, as well as in animal models (Musgrave et al., 2011; Paslawski, Treit,
Baker, George & Coutts, 1996). Female mice with EAE that were treated with PLZ were
observed to have decreased anxiety and increased exploratory behavior in an ‘open field’
behavioural assay (Musgrave et al., 2011). In this paradigm, the movement of mice were tracked
in an open field, and the number of times they crossed over the midline was recorded; increased
crossings suggest decreased anxiety and increased exploratory behaviours. PLZ-treated mice
showed significantly more crossings in the open field when compared to vehicle-treated EAE
animals, suggesting that PLZ beneficially decreases anxiety-like behaviour, resulting in
increased exploration (Musgrave et al., 2011). These effects are proposed to be a direct benefit of
increased GABA levels, which is supported by the evidence that anxiety was not reduced in the
elevated plus maze (EPM) when mice were treated with N2-Acetylphenelzine (N2-Ac-PLZ), a
derivative that has no elevating effect on GABA levels (Baker, Wong, Yeung & Coutts, 1991;
Musgrave et al., 2011; Paslawski et al., 1996). Increases of NA, 5-HT, and GABA levels via
PLZ administration have been demonstrated in both non-diseased, healthy animals as well as
mice in with EAE (Matveychuk et al., 2013). In the EAE model, these increases have been
accompanied by improved disease severity, delayed onset of the disease, and reduced mechanical
hypersensitivity in female mice with EAE (Benson et al., 2013; Musgrave et al., 2011; Potter et
al., 2016).
N2-Ac-PLZ, SEX DIFFERENCES & EAE 11
1.8 N2-Acetylphenelzine (N2-Ac-PLZ)
N2-Ac-PLZ is an acetylated derivative of PLZ that is also a non-selective inhibitor of
MAO’s. N2-Ac-PLZ acts to increase NA and 5-HT levels through inhibition of monoamine
oxidases, but dissimilar to PLZ, does not produce the active metabolite PEH, therefore does not
increase levels of GABA (McKenna, Baker & Coutts, 1991; McKenna, Baker, Coutts &
Greenshaw, 1992; Sowa et al., 2004). PLZ has been observed to have anxiolytic properties,
Figure 5. Molecular structure of N2-Ac-PLZ.
which were thought to be related to elevated GABA levels, and N2-Ac-PLZ has been reported to
have no such effect (Musgrave et al., 2011). These disparities between PLZ and N2-Ac-PLZ
provide the opportunity to study GABA’s mechanisms and importance in the production and
regulation of pain in EAE between the sexes.
1.9 Formalin: Effects of PLZ & N2-Ac-PLZ
The formalin test is a model of tonic pain processing that is commonly used to
examine nociceptive behaviour (Dubuisson & Dennis, 1977). Mice receive a subcutaneous
injection of formalin (37% formaldehyde solution in 0.9% saline) into the plantar surface of one
hindpaw and are then observed for 30 to 60 minutes while the time spent engaging in nociceptive
behaviours, such as licking, lifting, flinching of the injected paw, is recorded (Mifflin, Benson,
Thorburn, Baker & Kerr, 2016). Mifflin et al. (2016) observed that treatment with PLZ or N2-
Ac-PLZ prior to formalin significantly decreased nociceptive behaviours in male mice. The
N2-Ac-PLZ, SEX DIFFERENCES & EAE 12
response of female mice to PLZ, was however, less robust, and female mice were not responsive
at all to pre-treatment with N2-Ac-PLZ (Mifflin et al., 2016). Interestingly the levels of NA and
5-HT were increased to a similar level in both female and male spinal cords following pre-
treatment with N2-Ac-PLZ. These results suggest that MAO inhibitors, which act to increase
levels of NA and 5-HT, can significantly benefit nociception in males within a tonic pain model
but have mixed effects in females. Female mice were, however, responsive to pre-treatment with
PEH to a similar level as males, indicating that females may require GABA to control
nociception (Mifflin et al., 2016). The variance in nociceptive behavioural outcomes in
comparison to similar spinal levels of neurotransmitters offers an interesting sex difference that
should be explored further within a disease model with chronic pain.
1.10 Present Study: The Effects of N2-Ac-PLZ in the EAE Model of Multiple Sclerosis (MS)
The present study sought to investigate the potential therapeutic benefit of N2-Ac-PLZ
treatment on disease parameters and nociceptive behaviours in EAE. I also sought to determine
whether these effects were mediated by increased levels of NA and 5-HT in the CNS, and
whether there are sex-differences in the response to treatment with N2-Ac-PLZ. The animal
model EAE allowed us to ask several important questions regarding sex differences in EAE, as
well as examine the effects of an antidepressant derivative in a non-clinical setting.
In experiment 1, I examined the effects of N2-Ac-PLZ on nociceptive, exploratory,
and anxiety behaviours, as well as on the levels of NA, 5-HT, and GABA levels within the spinal
cord in non-diseased, naïve mice of both sexes. These observations are important in order to
understand the basal effects N2-Ac-PLZ in a non-diseased state. I predicted that treatment with
N2-Ac-PLZ will increase levels of NA and 5-HT equally between female and male mice. Based
N2-Ac-PLZ, SEX DIFFERENCES & EAE 13
on previous results from the formalin assay, I predicted that N2-Ac-PLZ will be more effective at
preventing pain hypersensitivity in naïve male mice.
In the next set of experiments, I assessed the effects of N2-Ac-PLZ on the disease
course of EAE, as well as nociceptive, exploratory, and anxiety behaviours, and spinal cord
neurotransmitter levels in both female and male mice with the disease. Based on results from the
experiments conducted with PLZ and N2-Ac-PLZ in the formalin model, I predicted that N2-Ac-
PLZ will increase NA and 5-HT levels equally in female and male mice with EAE, but only
reduce pain hypersensitivity in males with EAE.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 14
2. Methods
2.1 Subjects and Experimental Treatments. All mice (C57/BL6) were received from Charles
River Canada Laboratory, Canada. Mice were group-housed (5 mice per cage) in a controlled
room with a set 12-hour light/dark cycle throughout the entire experiment. All experimental
procedures were conducted during the light phase. Mice were given free access to standard food
pellets and water. All procedures were performed in compliance with the Canadian Council on
Animal Care Guidelines and Policies with the approval from the Animal Care and Use
Committee: Health Sciences for the University of Alberta (00000274). The information below
outlines the number of mice, age of mice, as well as procedures used for each experiment.
2.1-1 Naïve N2-Ac-PLZ Experiment
2.1-1.1 Female and male 6- to 8-week-old C57/BL6 mice were used for this experiment
(n= 72; 36 females, 36 males). On the testing days, mice underwent morning
baseline behavioural testing and then randomly divided into groups who received an
intraperitoneal injection of a vehicle treatment (bacteriostatic water), N2-Ac-PLZ
(39.82 mg/kg in bacteriostatic water; a dose calculated to be equivalent on a molar
basis to PLZ treatment at 30 mg/kg), or no injection at all (naïve). Mice were then
separately housed for three hours to allow for maximum effect of the injections
(McManus, Baker, Martin, Greenshaw & McKenna, 1992), then run through the
same behavioural tests (outlined below). N2-Ac-PLZ was obtained from the
laboratory of Dr. Glen Baker (Department of Psychiatry, University of Alberta,
Edmonton, Alberta, Canada).
2.1-1.2 Behavioural Testing. Prior to testing, all mice were left undisturbed for 2-3 days in
their home cages after arrival from Charles River Canada. This was done to allow
N2-Ac-PLZ, SEX DIFFERENCES & EAE 15
the animals to acclimatize to their new environment. Mice were also handled by the
experimenter for 3-5 days prior to baseline testing to ensure they were familiar with
the experimenter conducting the behavioural tests. Baseline testing was conducted
the week prior to any treatment. This two-week habituation period allowed the mice
to properly acclimatize to their environment, as well as ensuring that mice reached
sexual maturity, an important point to consider as potential sex differences were of
interest in these studies. The behavioural tests used in each experiment are outlined
below.
2.1-1.2.1 Exploratory Behaviour Testing. The open field test was used to assess
exploratory behaviours. A subset of mice (n=36; 18 females, 18 males) were
individually placed in an open field box and recorded to analyze behaviour.
Software was used to determine how much the animal explored the open field,
and how much time was spent in the center field (Ethovision version 9.0).
Mice underwent one baseline open field test a week before treatment to
minimize habituation, and then were tested once after drug treatment.
2.1-1.2.2 Anxiety Testing. The elevated plus maze (EPM) was used to measure anxiety
behaviour. A subset of mice (n= 36; 18 females, 18 males) was individually
placed in the center of a plus shaped apparatus, where two of the arms are
closed, and two of the arms are open. Time spent in open arms, as well as
number of entries into the open arms was measured, with increases in both
indicating decreased anxiety. Mice were tested only after treatment.
2.1-1.2.3 Mechanical Hypersensitivity Testing. Von-Frey hairs were used to measure
mechanical hypersensitivity, where the pain threshold was determined by the
N2-Ac-PLZ, SEX DIFFERENCES & EAE 16
lowest force (in grams) that caused a withdrawal response (i.e. shake, lift, or
lick) in three of five repetitive stimuli on the base of their hindlimb paws; a
lower threshold indicated increased hypersensitivity. A baseline test was
conducted in the morning prior to treatment and an experimental test was done
three hours after treatment for a subset of mice (n= 36; 18 females, 18 males).
2.1-1.2.4 Motor Function Testing. The rota-rod was used to measure gross locomotor
ability. A subset of mice (n= 36; 18 females, 18 males) were individually
placed on a rota-rod (a spinning rod) that was fixed at 12 rpm, and the
duration they were able to stay on was measured for three trials (up to 180 s)
and then averaged. The longer the mouse stays on the rod, the better the motor
function. A baseline test was conducted in the morning prior to treatment and
an experimental test was done three hours after treatment.
2.1-1.3 Tissue Collection Procedure. At the end of testing, all mice were euthanized via a
Euthansol (sodium phentobarbital) intraperitoneal injection and then transcardially
perfused with 0.9% saline. Whole spinal cord tissue samples were collected and
flash frozen in liquid nitrogen. Tissue was stored at -80°C prior to analysis.
2.1-1.4 High Performance Liquid Chromatography (HPLC). Samples were retrieved from
the -80°C freezer and kept on dry ice to prevent thawing, then weighed from all
experiments. Tissue was prepared for HPLC analysis to determine levels of amino
acids and biogenic amines following a protocol similar to Musgrave et al. (2011).
Amino Acids. To prepare samples for amino acids, the tissue was homogenized in 5
volumes of ice-cold water. An aliquot was added to 4 volumes of ice-cold methanol
and left on ice for 10 minutes, then centrifuged (12,000 g for 4 minutes). The
N2-Ac-PLZ, SEX DIFFERENCES & EAE 17
supernatant was diluted with distilled water to a final 120-fold dilution. Part of the
final supernatant was reacted with o-pthaldialdehyde and N-iso-butyryl-L-cysteine
dissolved in a borate buffer and resulting derivatives were used for analysis with a
fluorescence detector set at an excitation wavelength of 344 nm and an emission
wavelength of 433 m. Calibration curves for amino acids were prepared from
authentic amino acid samples and generated for each run of samples.
Biogenic Amines. To prepare samples for measurement of biogenic amines, aliquots
of tissues homogenized in 5 volumes of water were combined with 1/10th the
volume of ice-cold 1 M HClO4 containing ascorbic acid (500 m) and
ethylenediaminetetraacetic acid (100%/mg). Homogenates were vortexed and
centrifuged at 12,000g for 4 minutes. Subsequent supernatants were used for HPLC
analysis using electrochemical detection. Applied potential for electrochemical
detection was 0.65 V, and calibration curves were constructed for each point. All
water was distilled and purified by reverse osmosis using the Milli-Q filtration
system from Millipore (Billerica, Massachusetts). Methanol, tetrahydrofuran, and
acetonitrile were HPLC-grade obtained from Fisher Scientific (Pittsburgh, PA). All
solvents were filtered using Millipore nylon membranes (0.2-m pore size). o-
Pthaldialdehyde and ascorbic acid were from Sigma-Aldrich, N-iso-butyryl-L-
cysteine from NOVA chemicals (Calgary, Alberta, Canada) and sodium borate
from Fisher Scientific.
2.1-1.5 Statistical Analyses. Analyses were conducted using Sigmaplot software version
12.0 (Systat Software, Inc., San Jose, CA) using t-tests, one way ANOVAs, one
way repeated measure ANOVAS, or three way ANVOAs. Post-hoc analyses were
N2-Ac-PLZ, SEX DIFFERENCES & EAE 18
conducted if needed. If data failed to meet normal distribution criteria,
nonparametric tests were used where applicable. For all tests, significance was set
at P < 0.05.
2.1-2 Disease Related Chronic Pain Experiment.
2.1-2.1 Female and male 6- to 8-week-old C57/BL6 mice were used for this experiment
(n= 80; 40 females, 40 males).
EAE Induction. Mice in this experiment were given EAE to induce disease-related
chronic pain. It has previously been shown that this animal model of multiple
sclerosis is associated with chronic pain (Aharoni, 2013; Olechowski et al., 2009).
EAE is induced using myelin oligodendrocytes glycoprotein 35-55 (MOG35-55)
(Peptide Synthesis Facility, University of Calgary, Alberta, Canada). Forty-four
mice were induced with a 50 g subcutaneous injection of MOG35-55 emulsified in
Complete Freund’s Adjuvant (CFA) at a concentration of 1.5 mg/ml. Thirty-six
mice served as controls (CFA mice), and only received subcutaneous injections of
CFA. Both EAE and CFA mice received an intraperitoneal injection of 300 ng of
pertussis toxin (List Biological laboratories, Cedarlane, Canada) on the day of
induction and 48 h later. Mice were monitored daily (34 days post EAE induction)
for clinical signs of EAE and graded according to the following scale: Grade 0,
normal mouse; Grade 1, flaccid tail; Grade 2, mild hindlimb weakness with quick
righting reflex; Grade 3, severe hindlimb weakness with slow righting reflex; Grade
4, hindlimb paralysis in one hindlimb or both. This clinical scoring was used to
track the disease course of the animals, as well as the effectiveness of treatment
with N2-Ac-PLZ. To examine the effectiveness of N2-Ac-PLZ in reducing disease-
N2-Ac-PLZ, SEX DIFFERENCES & EAE 19
related chronic pain, EAE and CFA mice were divided randomly into drug
treatment groups. Mice received either an injection of vehicle treatment
(bacteriostatic water) or N2-Ac-PLZ (39.82 mg/kg in bacteriostatic water) every
other day. Injections began seven days post EAE induction.
2.1-2.2 Behavioural Testing. All testing followed the same methods as presented in Naïve
N2-Ac-PLZ experiment (2.1-1).
2.1-2.2.1 Exploratory Behaviour Testing. A subset of mice (n= 40; 20 females, 20
males) underwent three days of baseline testing, testing at onset of clinical
signs, and once per week for the duration of the experiment. Mice underwent
multiple testing time points in this experiment, as EAE and sickness in general
is known to reduce exploratory behaviour.
2.1-2.2.2 Mechanical Hypersensitivity Testing. Mice underwent three days of baseline
testing, testing at onset of clinical signs, and once per week for the duration of
the experiment.
2.1-2.2.3 Motor Function Testing. Mice underwent baseline testing, testing at onset, and
testing once per week.
2.1-2.3 Tissue Collection Procedure, HPLC, and Statistical Analyses. All followed the
same procedures as outlined in Naïve N2-Ac-PLZ experiment (2.1-1.3, 2.1-1.4,
2.1-1.5).
N2-Ac-PLZ, SEX DIFFERENCES & EAE 20
3. Results
3.1 Effects of N2-Ac-PLZ in Naïve Mice
To explore the baseline effects of N2-Ac-PLZ, I examined its effects after acute administration
in a variety of behavioural assays, as well as its influence on neurotransmitter levels in naïve
mice of both sexes.
3.1-1 Exploratory Behavior in the Open Field Test. I used the open field test to examine
exploratory behaviour in naïve female and male mice who were pretreated with vehicle, N2-
Ac-PLZ, or no injection at all. After the IP injection, mice were individually housed for 3
hours to allow for maximum effect of the treatment. The overall total distance moved around
the open field and time spent in the center zone of the field were analyzed for each treatment
group, and compared to a baseline level for both measures. Increased distance moved and
time spent in the center zone suggests that exploratory behavior is increased in these animals.
Our lab previously reported that PLZ increased the number of line crossings within the open
field test, indicating that exploratory behavior was increased, but suggested that these effects
were due to the increased GABA levels (Musgrave et al., 2011). I tested this theory by
treating naïve mice with N2-Ac-PLZ, which lacks the active metabolite PEH, therefore not
increasing GABA levels.
There was a significant effect in total distance moved in females (one-way ANOVA: F3,
35 = 6.119, P = 0.002; Fig 6A), with a significant decrease in distance moved from baseline to
no treatment (NT) and vehicle treated mice (Tukey’s post hoc, NT: q = 5.208, p = 0.005;
Veh: q = 4.300, p = 0.023). An effect was also found for total time spent in the center zone in
female mice (one-way ANOVA, H3 = 10.457, P = 0.015; Fig 6B). Non-treated female mice
were observed to spend significantly less time in the center when compared to baseline
N2-Ac-PLZ, SEX DIFFERENCES & EAE 21
(Dunn’s post hoc, Q = 2.778, p = 0.033). These results indicate that in general, exploratory
behaviour is decreased when compared to the baseline. These results may be due to any
stress the animals experience on the day of the experiment, including transport and being
housed individually after treatment, as well as habituation to the behavioural test and once
novel environment.
The exact same test was performed on males to observe any potential sex differences in
behaviour with N2-Ac-PLZ treatment. There was a significant effect in total distance moved
in male mice (one-way ANOVA, H3 = 12.945, P = 0.005; Fig 6C). Male mice treated with
vehicle were observed to move significantly less when compared to baseline levels (Dunn’s
post hoc, Q = 3.445, p = 0.003). A significant effect was also found for male time spent in
the center zone (one-way ANOVA, H3 = 12.620, P = 0.006; Fig 6D), where male NT mice
spent significantly less time in the center zone of the open field (Dunn’s post hoc, Q = 3.220,
p = 0.008). In general, males showed decreased total distance moved and time spent in the
center zone.
N2-Ac-PLZ treatment in females and males did not significantly increase total distance
moved or time spent in the center zone, indicating that it has no significant effects on
exploratory behaviour. These results support the hypothesis that Musgrave et al. (2011)
proposed, and suggest that improved exploratory behaviour may be regulated through a
GABAergic system.
3.1-2 Anxiety-like Behaviour in the Elevated Plus Maze. To determine if N2-Ac-PLZ could
beneficially reduce anxiety, I used the elevated plus maze (EPM) to evaluate any baseline
anxiolytic effects in naïve female and male mice. I used the number of entries into the open
arms, as well as time spent in the open arms, as outcome measures, with increased entries
N2-Ac-PLZ, SEX DIFFERENCES & EAE 22
and time indicating reduced anxiety. Analysis of the number of entries into the open arms of
both female and male mice as well as the time spent in open arms, did not reveal any
significant differences between non-treated control mice and those that were treated with
vehicle or N2-Ac-PLZ (Fig 7A-D). However there seems to be a trend towards normalization
of time spent in open arms for both sexes treated with N2-Ac-PLZ (Fig 7B,D).
3.1-3 Mechanical sensitivity in the Von-Frey Hair Test. To examine the effects of N2-Ac-
PLZ on baseline nociceptive sensitivity, I used the Von-Frey hair test in order to measure
responsiveness to mechanical stimuli. Stimulating the plantar surface of the hind paws, I used
stimuli with varying amounts of bending force in ascending order to elicit a response (i.e.
shake, lift, lick). The Von Frey filament that produced responses in at least three out of five
stimuli was deemed the ‘threshold’. I did not observe any changes in withdrawal thresholds
when compared to baseline levels in either sex (Fig 8A,B).
3.1-4 Motor Functioning in the Rota-Rod Test. The rota-rod test was used to determine
whether N2-Ac-PLZ had any effect on locomotor function. Analysis did not reveal any
significant differences in female or male mice between baseline and treatments (Fig 9A,B).
Both female and male mice remained on the rota-rod for the full duration of tests.
3.1-5 Levels of Neurotransmitters in the Spinal Cord. HPLC was used to assess the levels of
NA, 5-HT, and GABA within post-mortem spinal cord tissue. N2-Ac-PLZ has been reported
to increase NA and 5-HT levels due to its inhibitory effects on MAO’s, but does not affect
GABA levels (McKenna et al., 1991; McKenna et al., 1992; Sowa et al., 2004). All graphs
are expressed as a percent change from baseline. As expected, N2-Ac-PLZ treatment
significantly increased the levels of NA and 5-HT in female mice (t-test, U = 6.000, P<0.001,
Fig 10A; U = 0.000, P<0.001, Fig 10B) and in male mice (t-test, t(21) = -4.129, P = 0.0005;
N2-Ac-PLZ, SEX DIFFERENCES & EAE 23
Fig 10D; U = 4.000, P<0.001, Fig 10E). Changes in the levels of NA and 5-HT were notably
greater in female mice compared to males [NA: 113.25% (female) vs. 63.07% (male); 5-HT:
455.44% (female) vs. 214.60% (male)]. N2-Ac-PLZ had no effect on GABA levels in either
female or male mice (Fig 10C,F). These results provide evidence that N2-Ac-PLZ is
increasing levels of NA and 5-HT, but not affecting GABA levels.
3.2 Effects of N2-Ac-PLZ in Mice with EAE
To explore the potential beneficial effects of N2-Ac-PLZ in a chronic disease paradigm, I
administered N2-Ac-PLZ chronically to mice with EAE of both sexes and examined its effects
on disease parameters, behavioural assays, and neurotransmitter levels. CFA control mice or
mice with EAE were randomly sorted into treatment conditions, and either received vehicle or
N2-Ac-PLZ treatment every other day.
3.2-1 Disease Course and Day to Onset. Animals were monitored daily to assess their
clinical score according to the following scale: Grade 0, normal mouse; Grade 1, flaccid tail;
Grade 2, mild hindlimb weakness with quick righting reflex; Grade 3, severe hindlimb
weakness with slow righting reflex; Grade 4, hindlimb paralysis in one hindlimb or both.
This was done to monitor their clinical progression throughout the experiment and record
their day of disease onset (the day when they first present as clinical grade 1). Analysis did
not reveal any significant differences in female or male clinical progression (Fig 11A,C), or
day to onset with N2-Ac-PLZ treatment (Fig 11B,D), although the data indicate a trend
towards delayed onset for females (Fig 11B). In addition, it should be noted that male mice
had a significantly higher average peak clinical score than females, suggesting that disease
severity is greater in male mice than in females (t-test, U = 120.000, P = 0.018; Table 1).
N2-Ac-PLZ, SEX DIFFERENCES & EAE 24
3.2-2 Exploratory Behavior in the Open Field Test. Exploratory behavior was measured by
the total distance moved by female and male mice with EAE at onset (clinical grade 1), as
well as time spent in the center zone of the open field, and compared to baseline levels. There
was a significant reduction in the total distance moved in female mice with EAE (one-way
ANOVA, F2, 19 = 9.852, P = 0.001; Fig 12A). Total distance moved significantly decreased in
females treated with vehicle as well as N2-Ac-PLZ (Tukey post hoc, q = 4.637, p = 0.012; q
= 5.535, p = 0.003). In addition, N2-Ac-PLZ did not have any significant effects on the
amount of time spent in the center zone (Fig 12B).
Similarly, there was a significant reduction in the total distance moved by male mice
with EAE (one-way ANOVA, F2, 19 = 111.554, P < 0.001; Fig 12C). Distance traveled in the
open field was significantly decreased in male mice with EAE receiving either vehicle or N2-
Ac-PLZ when compared to baseline levels (Tukey post hoc, q = 17.083, p < 0.001; q =
17.409, p < 0.001). Unlike the female mice with EAE, there was a significant effect in the
amount of time that males spent in the center zone (one-way repeated measures ANOVA, F2,
19 = 28.195, P < 0.001; Fig 12D). Male mice with EAE who were treated with a vehicle or
N2-Ac-PLZ spent significantly less time in the center zone when compared to baseline
(Bonferroni post hoc, t = 6.949, p < 0.001; t = 5.940, p = 0.001).
In general, female and male mice with EAE moved significantly less, regardless of
treatment condition. The time spent in the center zone also decreased in females and males,
although this only reached statistical significance in male mice. These effects may be due to
habituation, stress, or increased nociceptive sensitivity the mice experienced during the
experiment (i.e. transport, housing, disease course).
N2-Ac-PLZ, SEX DIFFERENCES & EAE 25
3.2-3 Mechanical Hypersensitivity in the Von-Frey Hair Test. The Von-Frey hair task was
used to measure mechanical sensitivity in female and male CFA treated control mice and
mice with EAE. Withdrawal thresholds were determined by the force (grams) that elicited a
response (i.e. shake, lift, lick) in three of five mechanical stimuli. All graphs are expressed as
a percent change from baseline. N2-Ac-PLZ treatment significantly increased withdrawal
thresholds in female mice with EAE (t-test, t (13) = -2.355, P = 0.035; Fig 13B). Withdrawal
thresholds were normalized with N2-Ac-PLZ treatment compared to EAE mice treated with
vehicle. This decrease in withdrawal thresholds in vehicle-treated females suggests that these
EAE mice experienced allodynia (when a previously innocuous stimulus now elicits a painful
response), which has been previously reported (Olechowski et al., 2009). N2-Ac-PLZ
treatment did not have an effect on withdrawal thresholds in female CFA control mice
(although it should be noted that the data suggest a trend towards an increase in thresholds
when treated with N2-Ac-PLZ; Fig 13A). There were no significant differences in the
withdrawal thresholds of either male CFA control mice (Fig 13C) or male mice with EAE
that were treated with N2-Ac-PLZ (Fig 13D).
3.2-4 Motor Functioning in the Rota-Rod Test. The rota-rod test determines the locomotor
function of the mice, which is important when dealing with mice with EAE since their motor
abilities are affected later on in the disease course. However, motor function was not
significantly changed when assessed at disease onset (the time in which Von Frey hair testing
took place) and N2-Ac-PLZ treatment did not appear to have any effect on motor behaviour
in male mice from both the CFA or EAE groups (Fig 14A-D).
3.2-5 Levels of Neurotransmitters in the Spinal Cord. HPLC was used to measure
neurotransmitter levels in post-mortem spinal cord tissue. All graphs are expressed as a
N2-Ac-PLZ, SEX DIFFERENCES & EAE 26
percent difference from CFA vehicle controls. Previous data in naïve animals showed that
N2-Ac-PLZ increased NA and 5-HT levels in both female and male mice. I performed the
same HPLC analysis in EAE tissue to confirm that N2-Ac-PLZ increases these
neurotransmitters in a disease-related chronic pain paradigm.
A sex and drug interaction was found for NA levels (three-way ANOVA, F1, 57 =
4.258, P = 0.044; Fig 15A,D). N2-Ac-PLZ administration significantly increased NA levels
in female and male mice with CFA and EAE (Holm-Sidak post hoc, female: t = 6.789, p <
0.001, Fig 15B; male: t = 3.398, p = 0.001, Fig 15E). In general, females treated with N2-Ac-
PLZ had a larger increase of NA levels when compared to males (Holm-Sidak, t = 2.817, p =
0.007).
An interaction between group and drug was found for 5-HT levels in male and female
mice as well (three-way ANOVA, F1, 50 = 25.035, P < 0.001; Fig 15B,E). Treatment with N2-
Ac-PLZ acted to significantly increase 5-HT levels in both female and male mice with CFA
or EAE (Holm-Sidak post hoc, t = 10.100, p <0.001; t = 2.418, p = 0.020).
Surprisingly, a sex and drug interaction was found for GABA levels (three-way
ANOVA, F1, 55 = 4.499, P = 0.039; Fig 15C,F). Administration of N2-Ac-PLZ significantly
decreased GABA levels in female and CFA mice (Holm-Sidak post hoc, t = 2.492, p =
0.016). Additionally, GABA levels decreased in both female and male mice treated with a
vehicle (Holm-Sidak post hoc, t = 5.348, p < 0.001).
N2-Ac-PLZ, SEX DIFFERENCES & EAE 27
3.3 Figures
Figure 6. Exploratory behaviour measured using the open field test in naïve mice. Baseline (dark
gray bars) represents testing that was done a week before the treatments. None (light gray bars)
were the naïve mice that received no treatment (NT), vehicle (black bars) were the mice that
received a vehicle injection (bacteriostatic water), and N2-Ac-PLZ (white bars) were the mice
that received the injection of N2-Ac-PLZ on the experimental day. A) Total distance moved in
female mice. Non-treated and vehicle-treated female mice were observed to move significantly
less when compared to baseline levels. There was no effect on total distance moved in female
mice treated with N2-Ac-PLZ. B) Female time spent in the center zone of the open field. Non-
treated females spent significantly less time in the center zone compared to baseline levels.
Treatment with a vehicle or N2-Ac-PLZ had no effect. C) Total distance moved in male naïve
mice. Male mice who received the vehicle moved around significantly less when compared to
baseline levels. N2-Ac-PLZ had no significant effect on distance moved. D) Male time spent in
the center zone of the open field. Non-treated male mice spent significantly less time in the
center zone compared to baseline. *,#p<0.05, One-Way ANOVA followed by post hoc Tukey or
Dunn’s analysis. Data are means ± standard error of the mean (SEM).
N2-Ac-PLZ, SEX DIFFERENCES & EAE 28
Figure 7. Anxiety behaviour measured in naïve mice using the elevated plus maze (EPM). None
(light gray bars) represents the naïve mice that did not receive any treatment (NT), vehicle (black
bars) were the mice that received a vehicle injection, and N2-Ac-PLZ (white bars) were the mice
that received the N2-Ac-PLZ injection. The more entries and time spent in the open arms
indicates that the animals were less anxious. A, C) N2-Ac-PLZ treatment did not significantly
affect the number of entries into the open arms for females or males. B, D) N2-Ac-PLZ treatment
did not significantly increase the amount of time spent in the open arms in females or males.
Data are means ± SEM.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 29
Figure 8. Von-Frey hairs were used to assess mechanical sensitivity in naïve mice. Baseline
(dark gray bars) represents the responses that were recorded the morning of testing, vehicle
(black bars) were the mice that received a vehicle injection, and N2-Ac-PLZ (white bars) were
the mice that received the N2-Ac-PLZ injection. A, B) Treatment did not produce any significant
changes in response thresholds in either females or males. Data are means ± SEM.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 30
Figure 9. Naïve mice were tested on the rota-rod as a locomotor control measure. This ensures
that any differences in behaviour were due to treatment and not locomotor ability. Baseline (dark
gray bars) represents the responses that were recorded the morning of testing, vehicle (black
bars) were the mice that received a vehicle injection, and N2-Ac-PLZ (white bars) were the mice
that received the N2-Ac-PLZ injection. A, B) Treatment with a vehicle or N2-Ac-PLZ did not
impair motor function in females or males. Data are means ± SEM.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 31
Figure 10. Spinal levels of NA, 5-HT, and GABA were measured using high performance liquid
chromatography (HPLC) as compared to naïve (no treatment) controls. Black bars represent
naïve mice treated with a vehicle and white bars represent naïve mice treated with N2-Ac-PLZ.
All bars represent percent difference from naïve. A, D) Treatment with N2-Ac-PLZ significantly
increased female and male NA levels. B, E) Treatment with N2-Ac-PLZ significantly increased
female and male 5-HT levels. The effect of N2-Ac-PLZ on NA and 5-HT is greater in females
than males. C, F) Treatment with N2-Ac-PLZ did not significantly change female or male
GABA levels. *,#p<0.05, t-test. Data are means ± SEM.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 32
Table 1. Peak clinical scores that female and male mice with EAE reached, with the number 5
representing a mouse that died. *p<0.05, t-test.
Peak Scores
Female Male
5 4
4 4
4 4
4 4
4 4
1 4
4 4
3 3
4 3
4 4
3 4
1 5
4 4
2 5
3 5
3 5
4 5
4 4
5
3
4
4
Average 3.39 4.14
N2-Ac-PLZ, SEX DIFFERENCES & EAE 33
Figure 11. The effects of N2-Ac-PLZ on clinical scores of mice with EAE. Vehicle (black dots
and bars) represents the mice with EAE that were treated with vehicle injections every other day,
and N2-Ac-PLZ (white boxes and bars) were the mice with EAE that were treated with N2-Ac-
PLZ injections every other day. A, C) There was no significant difference between female or
male mice treated with vehicle or N2-Ac-PLZ. B, D) Day to onset was not significantly different
between female or male mice treated with either vehicle or N2-Ac-PLZ. Data are means ± SEM.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 34
Figure 12. Exploratory behaviour in mice with EAE measured using the open field test. Baseline
(dark gray bars) represents testing that was done before EAE induction, none (light gray bars)
were the naïve mice that received no treatment (NT), vehicle (black bars) were the mice that
received a vehicle injection (bacteriostatic water), and N2-Ac-PLZ (white bars) were the mice
that received the injection of N2-Ac-PLZ. A) Total distance moved in female mice with EAE.
Treatment with vehicle or N2-Ac-PLZ significantly decreased the total distance moved in the
open field test when compared to baseline. B) Time female mice with EAE spent in the center
zone of the open field. Treatment did not significantly affect females’ time spent in the center
zone. C) Total distance male mice with EAE moved. Vehicle or N2-Ac-PLZ treatment
significantly decreased the total distance moved when compared to baseline. D) Time male mice
with EAE spent in the center zone. Mice treated with vehicle or N2-Ac-PLZ spent significantly
less time in the center zone when compared to baseline times. *,#p<0.05, One-Way ANOVA or
One-Way Repeated Measures ANOVA followed by post hoc Tukey or Bonferroni analysis. Data
are means ± SEM.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 35
Figure 13. Von-Frey hairs were used to measure mechanical sensitivity in mice with EAE. CFA
and EAE mice received either vehicle (black bars) or N2-Ac-PLZ (white bars) treatment every
other day. All bars represent percent difference from naïve (CFA mice treated with vehicle). A,
C, D) Withdrawal thresholds in the Von-Frey test. N2-Ac-PLZ administration does not
significantly affect female CFA mice, or male CFA or EAE mice. B) Withdrawal thresholds to
Von Frey hair mechanical stimulation are significantly increased in female EAE mice after N2-
Ac-PLZ treatment when compared to vehicle-treated mice. *,#p<0.05, t-test. Data are means ±
SEM.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 36
Figure 14. Motor function in mice with EAE was measured using the rota-rod. A, B, C, D) No
motor deficits were observed with vehicle or N2-Ac-PLZ treatment in female or male CFA and
EAE mice. *,#p<0.05, One-Way ANOVA. Data are means ± SEM.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 37
Figure 15. Spinal levels of NA, 5-HT, and GABA in mice with EAE measured with HPLC.
Dark gray bars represent CFA mice treated with N2-Ac-PLZ, black bars represent EAE mice
treated with vehicle injections, and white bars represent EAE mice treated with N2-Ac-PLZ. All
bars represent percent difference from naïve (CFA mice treated with vehicle). A, D) NA levels in
female and male mice. N2-Ac-PLZ significantly increased NA levels in both female and male
CFA and EAE mice. Overall, females had a larger increase of NA levels with N2-Ac-PLZ
treatment than males. B, E) 5-HT levels in female and male mice. N2-Ac-PLZ treatment
significantly increased 5-HT levels in female and male CFA and EAE mice. C, F) GABA levels
in female and male mice. N2-Ac-PLZ treatment significantly decreased GABA levels in female
and male CFA mice when compared to vehicle treated CFA mice. Additionally, GABA levels
were decreased in female and male EAE mice treated with the vehicle, also when compared to
vehicle treated CFA mice (+). *,#,+p<0.05, Three-way ANOVA followed by post hoc Holm-
Sidak analysis. Data are means ± SEM.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 38
4. Discussion
4.1 The Present Study
The present study sought to examine the potential therapeutic effects of N2-Ac-PLZ in
the EAE model of MS. I assessed the effects of chronic N2-Ac-PLZ treatment on female and
male mice with EAE, and evaluated its effects on disease course and exploratory, anxiety-like,
and nociceptive behaviours, as well as important neurotransmitter levels within the spinal cord.
4.2 Disease Course
The observation that N2-Ac-PLZ did not significantly alter disease course in mice with
EAE of either sex suggests that GABA plays a stronger role in the disease than previously
indicated. Previous studies have shown that PLZ administration significantly improved disease
severity, and even delayed the onset of EAE in female mice (Benson et al., 2013; Musgrave et
al., 2011). To determine whether these beneficial effects on disease outcomes are mediated by
the changes in NA/5-HT or GABA I assessed the effects of N2-Ac-PLZ which only elevates the
levels of NA and 5-HT. N2-Ac-PLZ treatment did not however, significantly change disease
course in either females or males, suggesting that GABA plays a significant role in the
development and severity of EAE. I did observe a trend towards a delayed ‘day to onset’ in
female mice treated with N2-Ac-PLZ but this effect did not reach statistical significance.
Additionally, it should be noted that male mice with EAE had a more severe average peak
clinical score than females, suggesting that male’s disease was more severe than females. It is
important to realize that the roles of neurotransmitters are extremely complex. Even though
previous results seem to indicate that GABA is the primary component differing between PLZ
and N2-Ac-PLZ treatment, we should remember that there may be various factors, such as other
neurotransmitters and amino acids, that are playing a role in pain development and regulation.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 39
4.3 Exploratory and Anxiety Like Behaviour
The open field test was used to measure exploratory behaviour in mice. In general, total
distance moved and time spent in the center zone generally decreased in both naïve and EAE
mice after baseline. These results could be due to any number of reasons, but are most likely due
to habituation. The mice were placed in the apparatus more than once, therefore might have
become familiarized to the once novel environment. Additionally, increased stressors could also
be playing a role. The naïve mice received injections prior to testing, which is a stressor, then
were housed individually before being tested. This added stress could therefore be contributing
to the decreased distance moved and time spent in the center zone. The mice with EAE may have
experienced both of these stressors as well, but in addition, experienced the disease which could
have affected their desire/motivation to explore a new environment (Ennaceur, Michalikova, van
Rensburg & Chazot, 2006).
I used the elevated plus maze (EPM) to measure anxiety-like behaviour in naïve mice,
and observed similar results to Musgrave et al. (2011). N2-Ac-PLZ treatment did not
significantly increase the number of open arm entries or time spent in the open arms in either
sex. However, the data suggest that N2-Ac-PLZ may act to normalize time spent in open arms in
both females and males. This may indicate that although the number of entries has decreased,
overall the mice are spending more time in the open arms. These results are preliminary, and
were only observed in a subset of animals. These findings on the anxiolytic properties of N2-Ac-
PLZ in EAE-induced anxiety would need to be replicated in order to make firm conclusions.
4.4 Pain Behaviours: Mechanical Hypersensitivity
The observation that N2-Ac-PLZ treatment improved mechanical hypersensitivity in
female mice with EAE offers an opportunity to further explore the underlying mechanisms
N2-Ac-PLZ, SEX DIFFERENCES & EAE 40
behind sex differences in pain. Previous data have shown that PLZ treatment improves
mechanical hypersensitivity in female EAE mice (Potter et al., 2016). PLZ, and its derivative
N2-Ac-PLZ, have been shown to decrease time spent engaging in nociceptive behaviours in the
formalin model in male mice (Benson et al., 2013; Mifflin et al., 2016; Musgrave et al., 2011).
Whereas treatment with PEH, which only elevates levels of GABA decreased nociceptive
behaviours in both sexes (Mifflin et al., 2016). These findings suggest that GABA plays a strong
role in female nociception, whereas males can engage noradrenergic/serotonergic systems along
with GABA to modulate nociception.
It is important to note that the formalin model is only a tonic model of pain processing,
representing a completely different type of pain. It was important to explore the effects of N2-
Ac-PLZ in a model of disease-related chronic pain. In the present study, we found that N2-Ac-
PLZ-treated female mice with EAE had significantly higher thresholds in the Von-Frey task
when compared to vehicle-treated females at disease onset. The data also showed a trend towards
an increase in mechanical thresholds in female CFA control mice who received N2-Ac-PLZ.
These results are contrary to what we had initially predicted, based on data from the formalin
model, and the question that arises is why males did not benefit from N2-Ac-PLZ.
4.5 Neurotransmitter Levels
As predicted, N2-Ac-PLZ increased NA and 5-HT levels in naïve and diseased mice of
both sexes. Unexpectedly, naïve female mice had larger increases of NA and 5-HT levels when
compared to naïve males. Furthermore, female mice with EAE had higher levels of NA when
compared to males with EAE that were treated with N2-Ac-PLZ. These data did not support the
hypothesis that NA and 5-HT levels would be similarly elevated in both the sexes. It should be
noted that N2-Ac-PLZ increased levels of NA and 5-HT more in naïve mice than in mice with
N2-Ac-PLZ, SEX DIFFERENCES & EAE 41
EAE. The differences between naïve and EAE mice are likely due to increased stress and pain
from injections every other day, as well as coping with a disease that drastically decreases NA,
5-HT, and GABA levels (Musgrave et al., 2011). Therefore, N2-Ac-PLZ treatment may be
acting to normalize levels in mice with EAE, whereas it acts to significantly increase baseline
levels in naïve mice.
A surprising finding was that N2-Ac-PLZ treatment significantly decreased GABA levels
in CFA control female and male mice, although the decreases were small. Lowered GABA levels
have been reported as a characteristic of pain, so it is probable that these findings are a
consequence of the pain and stress of multiple injections (Jasmin et al., 2003; Zeilhofer, Möhler
& Lio, 2009). In general, GABA levels decreased in female and male mice with EAE, with no
significant difference between vehicle or N2-Ac-PLZ treated mice. Again, this is likely due to
the disease itself, since decreased levels of GABA have been correlated with EAE (Musgrave et
al., 2011; Musgrave et al., 2011). It is also possible that N2-Ac-PLZ may be affecting GABA
levels indirectly through primarily noradrenergic or serotonergic pathways (Attal et al., 2010;
Benson et al., 2015; Mico´, Ardid, Berrocoso & Eschalier, 2006; Dupuis et al., 2016; Eide &
Hole, 1993; Mika et al., 2013; Ortega, Fernández-Pastor, Callado & Meana, 2010).
4.6 Mechanism of Action
Although this study provides some interesting information regarding the effects of N2-
Ac-PLZ on nociception within females, I was unable to determine the specific mechanism of
action by which this happened. Studies have shown that pain develops and is regulated via
different neurotransmitter systems in each of the sexes, with GABA playing a significant role in
female nociceptive regulation compared to males who seem capable of engaging NA/5-HT and
GABA to regulate nociceptive sensitivity. Therefore, if N2-Ac-PLZ were to have antinociceptive
N2-Ac-PLZ, SEX DIFFERENCES & EAE 42
properties we would predict that it would primarily benefit male mice with EAE. Contrary to this
hypothesis, my results support the idea that female mice with EAE may not require a GABA
component to regulate nociception. N2-Ac-PLZ treatment improved tactile hypersensitivity in
female mice with EAE but was ineffective in males. Although my research focused on how NA,
5-HT and GABA levels were affected with N2-Ac-PLZ treatment, it is possible that other
neurotransmitters, such as dopamine (DA), were also affected and played a role in the observed
results (Jarcho, Mayer, Jiang, Feier & London, 2013). DA has been implicated in the modulation
of pain perception, and is supported by evidence demonstrating that drugs which enhance
dopaminergic neurotransmission provide analgesia in both rodents and a clinical population
(Baliki, Geha, Fields & Apkarian, 2011; Gerdelat-Mas et al., 2007; Jarcho et al., 2013; Pontieri,
Tanda & Di Chiara, 1995). Further investigation into N2-Ac-PLZ’s effects on other
neurotransmitters within the CNS would provide a better understanding of the mechanisms
underlying sex differences within pain. These findings suggest that the sex-specific mechanisms
that regulate pain behaviours are engaged differently depending on the type of pain (acute, sub
chronic and disease-related chronic).
4.6-1 Immune Response
A possible explanation for the results in the EAE model is differing immune responses of
female and male mice with EAE. Sex differences are apparent within MS, starting with the
majority of patients being female, and experiencing higher levels of neuropathic pain (Orton et
al., 2006). Female susceptibility to other autoimmune conditions is also elevated, and this is
suggested to be due to women’s strong immune response (Dunn et al., 2007; Whitacre, Reingold
& O’Looney, 1999). It has been observed that immune cell infiltration differs between the sexes
in both patients and animal models (Dunn et al., 2010; Dunn et al., 2007; Mifflin, Frieser,
N2-Ac-PLZ, SEX DIFFERENCES & EAE 43
Benson, Baker & Kerr, 2017; Sorge et al., 2015). It has been suggested that males use
microglial-dependent pathways for pain processing, whereas females preferentially use adaptive
immune cells but can switch to a glial-dependent mechanism if none are available (Sorge et al.,
2015). These differences may be due to the females having larger quantities of T-lymphocytes,
such as CD4+ and CD8+ cells, in both the periphery and spinal cord (Scotland, Stables, Madalli,
Watson & Gilroy, 2011). Another factor potentially contributing to these observed sex-
differences in immune function is a dissimilar expression of peroxisome proliferator activated
receptors (PPARs)α and γ in T-cells (Zhang et al., 2012). These different levels of PPAR and
PPAR in T cells in the sexes generate different populations of T-cells between the sexes. Male
mice have T cells that are skewed towards a Th17 phenotype (producing higher levels of IL-17)
while female T cells are skewed towards a Th1 phenotype (producing higher levels of IFN).
Whether these specific classes of T cells differentially affect pain sensitivity and/or the responses
to treatment is currently being investigated by other members of the Kerr laboratory.
It is worth noting that Benson et al. (2013) found that PLZ treatment did not affect female
mice’s immune responses in EAE. They measured infiltrating CD4+ T-cells and Iba-1 reactivity,
markers for inflammation and microglial/macrophage activation, in mice with EAE treated with
PLZ. PLZ had no significant effect on the number of CD4+ T-cells, or the density of Iba-1 in the
disease although they reported a delayed disease onset and improved outcomes with PLZ
treatment. Since no changes were observed in these immune parameters, it is possible that PLZ
could be modulating the phenotype or cytokine profile of the T-cells and/or infiltrating
macrophages, therefore improving the disease course and pain hypersensitivity. Other data have
shown that increased GABAergic activity improves disease severity in mice with EAE, but also
does not directly affect T-cells (Bhat et al., 2010). Based on these data, we can postulate that N2-
N2-Ac-PLZ, SEX DIFFERENCES & EAE 44
Ac-PLZ will not significantly affect the immune response (recruitment or density of cells in the
CNS) in either sex in EAE. However, it remains to be determined whether N2-Ac-PLZ can
influence the cytokine profile of activated T cells or macrophages, and whether it would impact
pain signaling in the disease. If N2-Ac-PLZ can modulate T-cell/macrophage phenotypes, then
one would predict that female T-cells and macrophages are more susceptible to this modulation.
4.6-2 Oxidative Stress & Pain
Oxidative stress (OS) may explain the ineffectiveness of N2-Ac-PLZ on hypersensitivity
in male mice with EAE. OS is reported to play an important role in many conditions such as MS
and chronic pain, although data vary. OS is classified as the imbalance between the production of
reactive oxygen species (ROS) and the cell’s ability to defend against them (Gilgun-Sherki,
Melamed & Offen, 2004). Increased OS carries consequences, such as increasing the CNS’s
susceptibility to neurodegeneration (Halliwell, 2001). OS is typically controlled by endogenous
antioxidant systems, but unregulated ROS generation can disrupt this essential balance (Valério
et al., 2009). ROS have been named as a major player in the development of MS, where elevated
ROS contributes to invoking an inflammatory response, and increasing pain (Valério et al.,
2009). This results in an influx of T cells and macrophages into the CNS which causes
considerable damage (Uttara, Singh, Zamboni & Mahajan, 2009). Data has shown that
decreasing levels of OS, or inhibiting ROS, in the spinal cord can produce anti-nociceptive
effects, and this has become a target in treatment development (Fidanboylu, Griffiths & Flatters,
2011; Valério et al., 2009; Wang et al., 2004). Studies within a clinical population have
suggested that oxidative damage, alongside an enhanced immune response, contribute to disease
progression in MS patients (Choi, Lee, Hughes, Denney & Lynch, 2016).
N2-Ac-PLZ, SEX DIFFERENCES & EAE 45
There are various means to measure OS, but commonly used ones include using the ratio
of gluthathione (GSH) and glutathione disulfide (GSSG) levels and/or the levels of the enzyme
superoxide dismutase (SOD) (Choi, Lee, Hughes, Denney & Lynch, 2016). GSH is a critical
endogenous antioxidant that acts to protect cells against oxidative damage by detoxifying ROS
(Bains & Shaw, 1997; Pompella, Visvikis, Paolicchi, De Tata & Casini, 2003). During
detoxification, GSH is converted to its oxidized form (GSSG), therefore as GSSG levels
increase, GSH/GSSG ratios decreased, and indicating increased OS (Choi, Lee, Hughes, Denney
& Lynch, 2016; Halliwell, 2001). Therefore, when GSSG levels increase, this leads to a reduced
GSH/GSSG ratio, and is indicative of increased OS (Lew, Pyke & Quintanilha, 1985; Schafer &
Buettner, 2001). SOD, another commonly used marker of OS, plays a role in the conversion of
superoxide (O2-) radicals into oxygen (O2) or hydrogen peroxide (H2O2). Decreased SOD activity
is indicative of increased OS (Zargari et al., 2007).
Within the EAE model, studies have shown that OS and damage differ between females
and males. Benson et al. (2015) observed that female mice with EAE who were offered
voluntary wheel running (exercise has been reported as a beneficial treatment in MS and EAE)
had an elevated GSH/GSSG ratio, indicating lower levels of OS. Previous data have also shown
that exercise increased GSH/GSSG ratios in male mice with EAE at a chronic time point (Mifflin
et al., 2017). In general, SOD levels and activity have also been found to decrease in EAE
(Miller, Walczak, Majsterek & Kędziora, 2013). Mifflin et al. (2017) observed reduced SOD
enzyme activity in diseased females, indicating that perhaps females with EAE are unable to use
this antioxidant pathway, or resources are used up and exhausted. The results produced by
Mifflin et al. (2017) were also accompanied by improvements in clinical disease in both females
and males, although unequally. These studies give a little insight into the research that is being
N2-Ac-PLZ, SEX DIFFERENCES & EAE 46
conducted regarding sex differences in OS and show us that even though there are obvious
differences between females and males, there are still a lot of questions that remain unanswered.
It is imperative that ratios of GSH/GSSG are examined in future research, in order to determine
if OS is counteracting any beneficial aspects of N2-Ac-PLZ.
4.7 GABA Decreases
I was unable to determine why GABA levels were generally decreasing, and why N2-Ac-
PLZ treatment further decreased these levels in CFA animals, although decreases were small. A
possible explanation for decreased GABA levels is that lowered levels have been identified as a
trademark of pain, and these mice are generally experiencing pain within this disease model no
matter the treatment they are receiving (Jasmin et al., 2003; Zeilhofer, Möhler & Lio, 2009). This
may also explain why GABA levels dropped in N2-Ac-PLZ-treated CFA mice. Although the
CFA mice did not receive the active EAE induction, they still received injections every other
day, and experienced the same stressors as the sick mice, which could contribute to lowered
GABA levels (Biggio et al., 1984).
Conclusion
While my study did not provide mechanistic insight on the therapeutic effects of N2-Ac-
PLZ, it did offer supporting evidence of sex differences within the EAE model. I have shown
that N2-Ac-PLZ effectively decreases mechanical hypersensitivity in female mice at the onset of
EAE. Interestingly, I showed that N2-Ac-PLZ acts to increase levels of NA and 5-HT in both
female and male spinal cords post-mortem, although not equally as was predicted. The use of the
EAE model provides a useful platform in examining the effects of potential therapeutic agents in
both disease course and nociceptive behaviours. It is also valuable in determining sex-specific
pathways in which hypersensitivity and disease outcome are regulated. Future investigation into
N2-Ac-PLZ, SEX DIFFERENCES & EAE 47
sex differences and the underlying mechanisms that regulate mechanical hypersensitivity within
a disease state are important next steps towards tailoring treatments for more efficacious pain
relief.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 48
References
Aharoni, R. (2013). New findings and old controversies in the research of multiple sclerosis and
its model experimental autoimmune encephalomyelitis. Expert Review of Clinical
Immunology, 9, 423-440.
Al-Nuaimi, S.K., MacKenzie, E.M., & Baker, G.B. (2012). Monoamine oxidase inhibitors and
neuroprotection: A review. American Journal of Therapeutics, 19, 436-448.
Andersen, A. M. (1975). Crystal and molecular structure of noradrenaline. Acta Chemica
Scandinavica B, 29, 871-876.
Anjaneyulu, M., & Chopra, K. (2004). Fluoxetine attenuates thermal hyperalgesia through 5
HT1/2 receptors in streptozotocin-induced diabetic mice. European Journal of
Pharmacology, 497, 285-292.
Ardid, D., Alloui, A., Brousse, G., Jourdan, D., Picard, P., Dubray, C., & Eschalier, A. (2001).
Potentiation of the antinociceptive effect of clomipramine by a 5-HT1A antagonist in
neuropathic pain in rats. British Journal of Pharmacology, 132, 1118-1126.
Ardid, D., Jourdan, D., Mestre, C., Villanueva, L., Le Bars, D., & Eschalier, A. (1995).
Involvement of bulbospinal pathways in the antinociceptive effect of clomipramine in the
rat. Brain Research, 695 (2), 253-256.
Attal, N., Cruccu, G., Baron, R., Haanpää, M., Hansson, P., Jensen, T.S., Nurmikko, T.;
European Federation of Neurological Societies. (2010). EFNS guidelines on the
pharmacological treatment of neuropathic pain: 2010 revision. European Journal of
Neurology, 17, 1113-1188.
Bains, J.S., & Shaw, C.A. (1997). Neurodegenerative disorders in humans: The role of
glutathione in oxidative stress-mediated neuronal death. Brain Research Reviews, 25,
335-358.
Baker, G.B., Wong, J.T., Yeung, J.M., & Coutts, R.T. (1991). Effects of the antidepressant
phenelzine on brain levels of gamma-aminobutyric acid (GABA). Journal of Affective
Disorders, 21, 201-211.
Baliki, M.N., Geha, P.Y., Fields, H.L., & Apkarian, A.V. (2011). Predicting value of pain and
analgesia: Nucleus accumbens response to noxious stimuli changes in the presence of
chronic pain. Neuron, 66, 149-160.
Benson, C., Mifflin, K., Kerr, B., Jesudasan, S.J., Dursun, S., & Baker, G. (2015). Biogenic
amines and the amino acids GABA and glutamate: Relationships with pain and
depression. Modern Trends in Pharmacopsychiatry, 30, 67-79. doi: 10.1159/000435933.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 49
Benson, C., Paylor, J.W., Tenorio, G., Winship, I., Baker, G., & Kerr, B.J. (2015). Voluntary
wheel running delays disease onset and reduces pain hypersensitivity in early
experimental autoimmune encephalomyelitis (EAE). Experimental Neurology, 271, 279-
290.
Benson, C.A., Wong, G., Tenorio, G., Baker, G.B., & Kerr, B.J. (2013). The MAO inhibitor
phenelzine can improve functional outcomes in mice with established clinical signs in
experimental autoimmune encephalomyelitis (EAE). Behavioural Brain Research, 252,
302-311.
Bhat, R., Axtell, R., Mitra, A., Miranda, M., Lock, C., Tsien, R.W., & Steinman, L. (2010).
Inhibitory role for GABA in autoimmune inflammation. Proceedings of the National
Academy of Sciences of the United States of America, 107, 2580-2585.
Biggio, G., Concas, A., Serra, M., Salis, M., Corda, M.G., Nurchi, V., Crisponi, C., & Gessa,
G.L. (1984). Stress and beta-carbolines decrease the density of low affinity GABA
binding sites; An effect reversed by diazepam. Brain Research, 305, 13-18.
Bittner, S., Afzali, A.M., Wiendl, H., & Meuth, S.G. (2014) Myelin oligodendrocyte
glycoprotein (MOG35-55) induced experimental autoimmune encephalomyelitis (EAE) in
C57BL/6 mice. Journal of Visualized Experiments, 86, doi:10.3791/51275.
Bormann, J. (2000). The ‘ABC’ of GABA receptors. Trends in Pharmacological Sciences, 21,
16-19.
Bormann, J., & Feigenspan, A. (1995). GABAC receptors. Trends in Neurosciences, 18, 515-519.
Bove, R., & Chitnis, T. (2014). The role of gender and sex hormones in determining the onset
and outcome of multiple sclerosis. Multiple Sclerosis, 20, 520-526.
Breivik, H., Collett, B., Ventafridda, V., Cohen, R., & Gallacher, D. (2006). Survey of chronic
pain in Europe: Prevalence, impact on daily life, and treatment. European Journal of
Pain, 10, 287-333.
Chebib, M., & Johnston, G.A.R. (1999). The ‘ABC’ of GABA receptors: A brief review.
Clinical and Experimental Pharmacology & Physiology, 26, 937-940.
Choi, I.Y., Lee, P., Hughes, A.J., Denney, D.R, & Lynch, S.G. (2016). Longitudinal changes of
cerebral glutathione (GSH) levels associated with the clinical course of disease
progression in patients with secondary progressive multiple sclerosis. Multiple Sclerosis
Journal. doi: 10.1177/1352458516669441.
Compston, A., & Coles, A. (2008). Multiple sclerosis. Lancet, 372, 1502-1517.
Cooper, J.R., Bloom, F.E., & Roth, H.R. (2003). The biochemical basis of neuropharmacology
(8th ed.). Oxford, United Kingdom: Oxford University Press.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 50
Dansie, E.J., & Turk, D.C. (2013). Assessment of patients with chronic pain. British Journal of
Anaesthesia, 111, 19-25.
Dray, A. (1995). Inflammatory mediators of pain. British Journal of Anaesthesia, 75, 125-131.
Dubuisson, D., & Dennis, S.G. (1977). The formalin test: A quantitative study of the
analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats.
Pain, 4, 161-174.
Dunn, S.E., Bhat, R., Straus, D.S., Sobel, R.A., Axtell, R., Johnson, A., Nguyen, K., Mukundan,
L., Moshkova, M., Dugas, J.C., Chawla, A., & Steinman, L. (2010). Peroxisome
proliferator-activated receptor δ limits the expansion of pathogenic Th cells during
central nervous system autoimmunity. The Journal of Experimental Medicine, 207, 1599-
1608.
Dunn, S.E., Ousman, S.S., Sobel, R.A., Zuniga, L., Baranzini, S.E., Youssef, S., Crowell, A.,
Loh, J., Oksenberg, J., & Steinman, L. (2007). Peroxisome proliferator-activated receptor
(PPAR)α expression in T cells mediates gender differences in development of T cell
mediated autoimmunity. The Journal of Experimental Medicine, 204, 321-330.
Dupuis, A., Wattiez A.S., Pinguet J., Richard D., Libert F., Chalus M., Aissouni Y., Sion B.,
Ardid D., Marin P., Eschalier A., & Courteix C. (2016). Increasing spinal 5HT2A
receptor responsiveness mediates anti-allodynic effect and potentiates fluoxetine efficacy
in neuropathic rats. Evidence for GABA release. Pharmacological Research. Advance
online publication. doi:10.1016/j.phrs.2016.09.021.
Dworkin, R.H., O’Connor, A.B., Backonja, M., Farrar, J.T., Finnerup, N.B., Jensen, T. S.,
Kalso, E.A., Loeser, J.D., Miaskowski, C., Nurmikko, T.J., Portenoy, R.K., Rice, A.S.,
Stacey, B.R., Treede, R.D., Turk, D.C., & Wallace, M.S. (2007). Pharmacologic
management of neuropathic pain: Evidence-based recommendations. Pain, 132, 237-
251.
Eide, P.K., & Hole, K. (1993). The role of 5-hydroxytryptamine (5-HT) receptor subtypes and
plasticity in the 5-HT systems in the regulation of nociceptive sensitivity. Cephalalgia,
13, 75-85.
Elliott, A.M., Smith, B.H., Penny, K.I., Smith, W.C., & Chambers, W.A. (1999). The
epidemiology of chronic pain in the community. The Lancet, 354, 1248- 1252.
Enna, S.J., & McCarson, K.E. (2006). The role of GABA in the mediation and perception of
pain. Advances in Pharmacology, 54, 1-27.
Ennaceur, A., Michalikova, S., van Rensburg, R., & Chazot, P.L. (2006). Models of anxiety:
responses of mice to novelty and open spaces in a 3D maze. Behavioural Brain Research,
174, 9-38.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 51
Fidanboylu, M., Griffiths, L.A., & Flatters, S.J. (2011). Global inhibition of reactive oxygen
species (ROS) inhibits paclitaxel-induced painful peripheral neuropathy. PLoS One, 6,
doi: 10.1371/journal.pone.0025212.
Gendreau, R.M., Thorn, M.D., Gendreau, J.F., Kranzier, J.D., Ribeiro, S., Gracely, R.H.,
Williams, D.A., Mease, P.J., McLean, S.A., & Clauw, D.J. (2005). Efficacy of
milnacipran in patients with fibromyalgia. The Journal of Rheumatology, 32, 1975-1985.
Gerdelat-Mas, A., Simonette-Moreau, M., Talamas, C., Ory-Magne, F., Slaoui, T., Rascol, O., &
Brefel-Courbon, C. (2007). Levodopa raises objective pain threshold in Parkinson’s
disease: A RIII reflex study. Journal of Neurology, Neurosurgery & Psychiatry, 78,
1140-1142.
Gilgun-Sherki, Y., Melamed, E., & Offen, D. (2004). The role of oxidative stress in the
pathogenesis of multiple sclerosis: The need for effective antioxidant therapy. Journal of
Neurology, 251, 261-268.
Halliwell, B. (2001). Role of free radicals in the neurodegenerative diseases: Therapeutic
implications for antioxidant treatment. Drugs & Aging, 18, 685-716.
Hannon, J., & Hoyer, D. (2008). Molecular biology of 5-HT receptors. Behavioural Brain
Research, 195, 198-213.
Hofstetter, H.H., Shive, C.L., & Forsthuber, T.G. (2002). Pertussis toxin modulates the
immune response to neuroantigens injected in incomplete Freund’s adjuvant:
Induction of Th1 cells and experimental autoimmune encephalomyelitis in the
presence of high frequencies of Th2 cells. Journal of Immunology, 169, 117-125.
Jarcho, J.M., Mayer, E.A., Jiang, K., Feier, N., & London, E.D. (2013). Pain, affective symptoms
and cognitive deficits in patients with cerebral dopamine dysfunction. Pain, 153,
doi: 10.1016/j.pain.2012.01.002.
Jasmin, L., Rabkin, S.D., Granato, A., Boudah, A., & Ohara, P.T. (2003). Analgesia and
hyperalgesia from GABA-mediated modulation of the cerebral cortex. Nature, 424,
316-320.
Jasmin, L., Wu, M.V., & Ohara, P.T. (2004). GABA puts a stop to pain. Current Drug Targets.
CNS and Neurological Disorders, 3, 487-505.
Johnston, J.P. (1968) Some observations upon a new inhibitor of monoamine oxidase in brain
tissue. Biochemical Pharmacology, 17, 1285-1297.
Knoll, J., & Magyar, K. (1972). Some puzzling pharmacological effects of monoamine oxidase
inhibitors. Advances in Biochemical Psychopharmacology, 5, 393-408.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 52
Lew, H., Pyke, S., & Quintanilha, A. (1985). Changes in the glutathione status of plasma, liver
and muscle following exhaustive exercise in rats. FEBS Letters, 185, 262-266.
Linthicum, D.S., Munoz, J.J. & Blaskett, A. (1982). Acute experimental autoimmune
encephalomyelitis in mice. I. Adjuvant action of Bordetella pertussis is due to vasoactive
amine sensitization and increased vascular permeability of the central nervous system.
Cellular Immunology, 73, 299-310.
Liu, M.Y., Su. C.F., & Lin, M.T. (1988). The antinociceptive role of a bulbospinal serotonergic
pathway in the rat brain. Pain, 33, 123-129.
MacKenzie, E.M., Song, M.S., Dursun, S.M., Tomlinson, S., Todd, K.G., & Baker, G. B. (2010).
Phenelzine: an old drug that may hold clues to the development of new neuroprotective
agents. Bulletin of Clinical Psychopharmacology, 20, 179-186.
Marks, D.M., Shah, M.J., Patkar, A.A., Masand, P.S., Park, G.Y., & Pae, C.U. (2009). Serotonin
norepinephrine reuptake inhibitors for pain control: Premise and promise. Current
Neuropharmacology, 7, 331-336.
Matveychuk, D., Nunes, E., Ullah, N., Velázquez-Martinez, C.A., MacKenzie, E.M., & Baker,
G.B. (2013). Comparison of phenelzine and geometric isomers of its active metabolite, β
phenylethylidenehydrazine, on rat brain levels of amino acids, biogenic amine
neurotransmitters and methylamine. Journal of Neural Transmission, 120, 987-996.
McKenna, K.F., Baker, G.B., & Coutts, R.T. (1991). N2-Acetylphenelzine: Effects on rat brain
GABA, alanine and biogenic amines. Naunyn-Schmiedeberg's Archives of
Pharmacology, 343, 478-482.
McKenna, K.F., Baker, G.B., Coutts, R.T., & Greenshaw, A.J. (1992). Chronic administration of
the antidepressant-antipanic drug phenelzine and its N-acetylated analogue: Effects on
monoamine oxidase, biogenic amines, and alpha 2-adrenoceptor function. Journal of
Pharmaceutical Sciences, 81, 832-835.
McManus, D.J., Baker, G.B., Martin, I.L., Greenshaw, A.J., & McKenna, K.F. (1992). Effects of
the antidepressant/antipanic drug phenelzine on GABA concentrations and GABA
transaminase activity in rat brain. Biochemical Pharmacology, 43, 2486-2489.
Melzack, R., & Wall, P.D. (1965). Pain mechanisms: A new theory. Science, 150, 971-979.
Merskey, H. & Bogduk, N. (2011). Classification of chronic pain (2nd ed.). Seattle: IASP Press,
retrieved from
www.iasppain.org/files/Content/ContentFolders/Publications2/Classificationsof
ChronicPain/Part_III-PainTerms.pdf
Mico´, J.A., Ardid, D., Berrocoso, E., & Eschalier, A. (2006). Antidepressants and pain. Trends
in Pharmacological Sciences, 27, 348-354.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 53
Mifflin, K.A., Frieser, E., Benson, C., Baker, G., & Kerr, B.J. (2017). Voluntary wheel running
differentially affects disease outcomes in male and female mice with experimental
autoimmune encephalomyelitis. Journal of Neuroimmunology, 305, 135-144.
Mifflin, K.A., & Kerr, B.J. (2016). Pain in autoimmune disorders. Journal of Neuroscience
Research. doi: 10.1002/jnr.23844.
Mika, J., Zychowska, M., Makuch, W., Rojewska, E., & Przewlocka, B. (2013). Neuronal and
immunological basis of action of antidepressants in chronic pain- clinical and
experimental studies. Pharmacological Reports, 65, 1611-1621.
Millan, M.J. (1995). Serotonin (5-HT) and pain: A reappraisal of its role in the light of receptor
multiplicity. Seminars in the Neurosciences, 7, 409-419
Miller, E., Walczak, A., Majsterek. I., & Kędziora, J. (2013). Melatonin reduces oxidative stress
in the erythrocytes of multiple sclerosis patients with secondary progressive clinical
course. Journal of Neuroimmunology, 257, 97-101.
Möhler, H. (2012). The GABA system in anxiety and depression and its therapeutic potential.
Neuropharmacology, 62, 42-53.
Muñoz-Islas, E., Vidal-Cantú, G.C., Bravo-Hernández, M., Cervantes-Durán, C., Quiñonez
Bastidas, G.N., Pineda-Farias, J.B., Barragán-Iglesias, P., & Granados Soto, V. (2014).
Spinal 5-HT5A receptors mediate 5-HT-induced antinociception in several pain models in
rats. Pharmacology, Biochemistry and Behaviour, 120, 25-32.
Musgrave, T., Benson, C., Wong, G., Browne, I., Tenorio, G., Rauw, G., Baker, G.B., & Kerr,
B. J. (2011). The MAO inhibitor phenelzine improves functional outcomes in mice with
experimental autoimmune encephalomyelitis (EAE). Brain, Behaviour, and Immunity, 25,
1677-1688.
Musgrave, T., Tenorio, G., Rauw, G., Baker, G.B., & Kerr, B.J. (2011). Tissue concentration
changes of amino acids and biogenic amines in the central nervous system of mice with
experimental autoimmune encephalomyelitis (EAE). Neurochemistry International, 59,
28-38.
Nalepa, I., Vetulani, J., Borghi, V., Kowalska, M., Przewłocka, B. & Pavone, F. (2005).
Formalin hindpaw injection induces changes in the [3H]prazosin binding to 1
adrenoceptors in specific regions of the mouse brain and spinal cord. Journal of Neural
Transmission, 112, 1309-1319.
Okamoto, K., Imbe, H., Kimura, A., Donishi, T., Tamai, Y., & Senba, E. (2007). Activation of
central 5HT2A receptors reduces the craniofacial nociception of rats. Neuroscience, 147,
1090-1102.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 54
Olechowski, C.J., Truong, J.J., & Kerr, B.J. (2009). Neuropathic pain behaviours in a chronic
relapsing model of experimental autoimmune encephalomyelitis (EAE). Pain, 141, 156
164.
Ortega, J.E., Fernández-Pastor, B., Callado, L.F., & Meana, J.J. (2010). In vivo potentiation of
reboxetine and citalopram effect on extracellular noradrenaline in rat brain by 𝛼2
adrenoceptor antagonism. European Neuropsychopharmacology, 20, 813-822.
Orton, S.M., Herrera, B.M., Yee, I.M., Valdar, W., Ramagopalan, S.V., Sadovnick, A.D., Ebers,
G.C.; Canadian Collaborative Study Group. (2006). Sex ratio of multiple sclerosis in
Canada: A longitudinal study. The Lancet Neurology, 5, 932-936.
Parent, M.B., Habib, M.K., & Baker, G.B. (2000). Time-dependent changes in brain monoamine
oxidase activity and in brain levels of monoamines and amino acids following acute
administration of the antidepressant/antipanic drug phenelzine. Biochemical
Pharmacology, 59, 1253-1263.
Paslawski, T., Treit, D., Baker, G.B., George, M., & Coutts, R.T. (1996). The antidepressant
drug phenelzine produces antianxiety effects in the plus-maze and increases in rat brain
GABA. Psychopharmacology, 127, 19-24.
Peng, Y.B., Lin, Q., & Willis, W.D. (1996). The role of 5-HT3 receptors in periaqueductal gray
induced inhibition of nociceptive dorsal horn neurons in rats. Journal of Pharmacology
and Experimental Therapeutics, 276, 116-124.
Pertovaara, A. (2006). Noradrenergic pain modulation. Progress in Neurobiology, 80, 53-83.
Pertovaara, A. (2013). The noradrenergic pain regulation system: A potential target for pain
therapy. European Journal of Pharmacology, 716, 2-7.
Pöllmann, W., & Feneberg, W. (2008). Current management of pain associated with multiple
sclerosis. CNS Drugs, 22, 291-324.
Pompella, A., Visvikis, A., Paolicchi, A., De Tata, V., & Casini, A.F. (2003). The changing faces
of glutathione, a cellular protagonist. Biochemical Pharmacology, 66, 1499-1503.
Pontieri, F.E., Tanda, G., & Di Chiara, G. (1995). Intravenous cocaine, morphine, and
amphetamine preferentially increase extracellular dopamine in the "shell" as compared
with the "core" of the rat nucleus accumbens. Proceedings of the National Academy of
Sciences of the United States of America, 92, 12304-12308.
Potter, L.E., Paylor, J.W., Suh, J.S., Tenorio, G., Caliaperumal, J., Colbourne, F., Baker, G.,
Winship, I., & Kerr, B.J. (2016). Altered excitatory-inhibitory balance within
somatosensory cortex is associated with enhanced plasticity and pain sensitivity in a
mouse model of multiple sclerosis. Journal of Neuroinflammation, 13, doi:
10.1186/s12974-0160609-4.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 55
Robinson, A.P., Harp, C.T., Noronha, A., & Miller, S.D. (2014). The experimental autoimmune
encephalomyelitis (EAE) model of MS: Utility for understanding disease
pathophysiology and treatment. Handbook of Clinical Neurology, 122, 173-189.
Ruffolo, R.R. Jr., & Hieble, J.P. (1994). -Adrenoceptors. Pharmacology & Therapeutics, 61, 1-
64.
Sawynok, J. (2003). Topical and peripherally acting analgesics. Pharmacological Reviews, 55,
1-20.
Schafer, F.Q., & Buettner, G.R. (2001). Redox environment of the cell as viewed through the
redox state of the gluthatione disulfide/glutathione couple. Free Radical Biology and
Medicine, 30, 1191-1212.
Scotland, R.S., Stables, M.J., Madalli, S., Watson, P., & Gilroy, D.W. (2011). Sex differences in
resident immune cell phenotype underlie more efficient acute inflammatory responses in
female mice. Blood, 118, 5918-5927.
Shi, T.J., Winzer-Serhan, U., Leslie, F. & Hökfelt, T. (1999). Distribution of 2-adrenoceptor
mRNAs in the rat lumbar spinal cord in normal and axotomized rats. NeuroReport, 10,
2835-2839.
Shih, J.C., & Thompson, R.F. (1999). Monoamine oxidase in neuropsychiatry and behaviour.
American Journal of Human Genetics, 65, 593-598.
Sindrup, S.H., Otto, M., Finnerup, N.B., & Jensen, T.S. (2005). Antidepressants in the treatment
of neuropathic pain. Basic & Clinical Pharmacology & Toxicology, 96, 399-409.
Smith, A.J. (1998). The analgesic effects of selective serotonin reuptake inhibitors. Journal of
Psychopharmacology, 12, 407-413.
Solaro, C., Brichetto, G., Amato, M.P., Cocco, E., Colombo, B., D’Aleo, G., Gasperini, C.,
Ghezzi, A., Martinelli, V., Milanese, C., Patti, F., Trojano, M., Verdun, E., Mancardi,
G.L.; PaIMS Study Group. (2004). The prevalence of pain in multiple sclerosis: A
multicenter cross-sectional study. Neurology, 14, 919-921.
Solaro, C., Trabucco, E., & Messmer Uccelli, M. (2013). Pain and multiple sclerosis:
Pathophysiology and treatment. Current Neurology and Neuroscience Reports, 13, doi:
10.1007/s11910-012-0320-5.
Sorge, R.E., Mapplebeck, J.C., Rosen, S., Beggs, S., Taves, S., Alexander, J.K., Martin, L.J.,
Austin, J.S., Sotocinal, S.G., Chen, D., Yang, M., Shi, X.Q., Huang, H., Pillon, N.J.,
Bilan, P.J., Tu, Y., Klip, A., Ji, R.R., Zhang, J., Salter, M.W., & Mogil, J.S. (2015).
Different immune cells mediate mechanical pain hypersensitivity in male and female
mice. Nature Neuroscience, 18, 1081-1083.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 56
Sowa, B.N., Holt, A., Todd, K.G., & Baker, G.B. (2004). Monoamine oxidase inhibitors, their
structural analogues, and neuroprotection. Indian Journal of Experimental Biology, 42,
851-857.
Stahl, S.M., Grady, M.M., Moret, C., & Briley, M. (2005). SNRIs: Their pharmacology, clinical
efficacy, and tolerability in comparison with other classes of antidepressants. CNS
Spectrums, 10, 732-747.
Stamford, J.A. (1995). Descending control of pain. British Journal of Anaesthesia, 75, 217-227.
Ugarte, S.D., Homanics, G.E., Firestone, L.L., & Hammond, D.L. (2000) Sensory thresholds and
the antinociceptive effects of GABA receptor agonists in mice lacking the β3 subunit
of the GABA(A) receptor. Neuroscience, 95, 795–806.
Uttara, B., Singh, A.V., Zamboni, P., & Mahajan, R.T. (2009). Oxidative stress and
neurodegenerative diseases: A review of upstream and downstream antioxidant
therapeutic options. Current Neuropharmacology, 7, 65-74.
Valério, D.A., Georgetti, S.R., Magro, D.A., Casagrande, R., Cunha, T.M., Vicentini, F.T.,
Vieira, S.M., Fonseca, M.J., Ferreira, S.H., Cunha, F.Q., & Verri, W.A. Jr. (2009).
Quercetin reduces inflammatory pain: Inhibition of oxidative stress and cytokine
production. Journal of Natural Products, 72, 1975-1979.
Wang, Z.Q., Porreca, F., Cuzzocrea, S., Galen, K., Lightfoot, R., Masini, E., Muscoli, C.,
Mollace, V., Ndengele, M., Ischiropoulos, H., & Salvemini, D. (2004). A newly
identified role for superoxide in inflammatory pain. The Journal of Pharmacology and
Experimental Therapeutics, 309, 869-878.
Watanabe, M., Maemura, K., Kanbara, K., Tamayama, T., & Hayasaki, H. (2002). GABA and
GABA receptors in the central nervous system and other organs. International Review of
Cytology, 213, 1-47.
Westlund, K.N., & Coulter, J.D. (1980). Descending projections of the locus coeruleus and
subnoeruleus/medial parabrachial nuclei in monkey: Axonal transport studies and
dopamine-β-hydroxylase immunohistochemistry. Brain Research Reviews, 2, 235-
264.
What is MS? Does everyone experience MS the same way? (n.d.). Multiple Sclerosis Society of
Canada. Retrieved from https://mssociety.ca/about-ms/what-is-ms
Whitacre, C.C., Reingold, S.C., & O’Looney, P.A. (1999). A gender gap in autoimmunity.
Science, 283, 1277-1278.
Wood, P.L., Khan, M.A., Moskal, J.R., Todd, K.G., Tanay, V.A.M.I., & Baker, G. (2006).
Aldehyde load in ischemia-reperfusion brain injury: Neuroprotection by neutralization of
reactive aldehydes with phenelzine. Brain Research, 1122, 184-190.
N2-Ac-PLZ, SEX DIFFERENCES & EAE 57
Yaksh, T.L. (1989) Behavioral and autonomic correlates of the tactile evoked allodynia produced
by spinal glycine inhibition: effects of modulatory receptor systems and excitatory
amino acid antagonists. Pain 37, 111–123
Yaksh, T. L., & Malmberg, A. B. (1994). Central pharmacology of nociceptive transmission.
Textbook of Pain, 4, 371-414.
Yaksh, T.L., & Wilson, P.R. (1979). Spinal serotonin terminal system mediates antinociception.
Journal of Pharmacology and Experimental Therapeutics, 208, 446-453.
Zargari, M., Allameh, A., Sanati, M.H., Tiraihi, T., Lavasani, S., & Emadyan, O. (2007).
Relationship between the clinical scoring and demyelination in central nervous system
with total antioxidant capacity of plasma during experimental autoimmune
encephalomyelitis development in mice. Neuroscience Letters, 412, 24-28.
Zeilhofer, H.U. (2008). Loss of glycinergic and GABAergic inhibition in chronic pain
contributions of inflammation and microglia. International Immunopharmacology, 8,
182-187.
Zeilhofer, H.U., Möhler, H., & Di Lio, A. (2009). GABAergic analgesia: New insights from
mutant mice and subtype-selective agonists. Trends in Pharmacological Sciences, 30,
397-402.
Zhang, M.A., Rego, D., Moshkova, M., Kebir, H., Chruscinski, A., Nguyen, H., Akkermann, R.,
Stanczyk, F.Z., Prat, A., Steinman, L., & Dunn, S.E. (2012). Peroxisome proliferator
activated receptor (PPAR)α and –γ regulate IFN γ and IL-17A production by human T
cells in a sex-specific way. Proceedings of the National Academy of Sciences of the
United States of America, 109, 9505-9510.